Isoelectric focusing devices and fixtures

ABSTRACT

Methods, devices, and systems for performing isoelectric focusing reactions are described. The systems or devices disclosed herein may comprise fixtures that have a membrane. In some instances, the disclosed devices may be designed to perform isoelectric focusing or other separation reactions followed by further characterization of the separated analytes using mass spectrometry. The disclosed methods, devices, and systems provide for fast, accurate separation and characterization of protein analyte mixtures or other biological molecules by isoelectric point.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Non Provisionalpatent application Ser. No. 16/808,063, filed Mar. 3, 2020, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 62/909,675,filed Oct. 2, 2019, U.S. Provisional Patent Application Ser. No.62/893,549, filed Aug. 29, 2019, and U.S. Provisional Patent ApplicationSer. No. 62/885,733, filed Aug. 12, 2019, each of which applications isherein incorporated by reference in its entirety for all purposes.

BACKGROUND

This disclosure relates to methods, devices, and systems for sampleprocessing and characterization, and various uses thereof. In a firstaspect, this disclosure relates to methods, devices, and systems forperforming separation and characterization of analytes in a mixture ofanalytes, and more specifically to multichannel devices (and relatedmethods and systems) for performing multiple isoelectric focusingreactions in parallel. In a second aspect, this disclosure relates tomicrofluidic devices (and related methods and systems) designed toperform one or more separation reactions (e.g., isoelectric focusing)followed by mobilization and electrospray ionization of the separatedanalytes for characterization by mass spectrometry.

SUMMARY

Disclosed herein are methods, devices, and systems that enable improvedquantitative performance for the separation and analysis of analytes inan analyte mixture, with potential applications in biomedical research,clinical diagnostics, and pharmaceutical manufacturing. For example,rigorous characterization of biologic drugs and drug candidates (e.g.,proteins) are required by regulatory agencies. The methods and devicesdescribed herein may be suitable for characterizing proteins and/orother analytes. In some instances, the methods and devices describedherein may relate to characterizing an analyte mixture wherein one ormore enrichment steps are performed to separate an analyte mixture intoenriched analyte fractions. In some instances, the methods and devicesdescribed herein may relate to performing one or more enrichment stepsto separation an analyte mixture into enriched analyte fractions in amultiplexed format for high throughput characterization of samples. Insome instances, the methods and devices described herein relate tocharacterizing an analyte mixture wherein one or more enrichment stepsare performed to separate an analyte mixture into enriched analytefractions that are subsequently introduced into a mass spectrometer viaan electrospray ionization interface. The disclosed methods and devicesmay provide improvements in convenience, reproducibility, and/oranalytical performance of analyte separation and characterization.

In an aspect, disclosed herein is a fixture comprising: an electrodereservoir; an inlet fluid channel comprising a first end and a secondend; an outlet fluid channel comprising a first end that is fluidicallycoupled to the second end of the inlet fluid channel, and a second endthat is fluidically coupled to a separation channel, wherein the inletfluid channel and the outlet fluid channel intersect with and arefluidically coupled to each other at a plane that defines or is parallelto a surface of the electrode reservoir; and a membrane disposed withinthe electrode reservoir at or adjacent to the plane such that themembrane covers all or substantially all of an opening comprising theintersection of the inlet fluid channel and the outlet fluid channel;wherein the membrane provides a high hydrodynamic resistance, lowelectrical resistance connection between a high voltage electrodepositioned within the electrode reservoir and a fluid contained withinthe inlet fluid channel and outlet fluid channel.

In some embodiments, the membrane is hydrophilic. In some embodiments,the membrane comprises a regenerated cellulose membrane. In someembodiments, the membrane comprises a woven polytetrafluoroethylene(PTFE) membrane that has been treated to be hydrophilic. In someembodiments, a cross-sectional area of the membrane or opening isbetween about 0.001 mm² and 100 mm². In some embodiments, the electrodereservoir further comprises an insert disposed within the electrodereservoir and positioned at or adjacent to the membrane, wherein theinsert comprises an inlet fluid path and an outlet fluid path thatfacilitate substantially bubble-free wetting of a surface of themembrane when the electrode reservoir is filled with an electrolytesolution.

In some embodiments, the hydrodynamic resistance between the electrodereservoir and the intersection of the inlet fluid channel and outletfluid channel is greater than 0.1 ((N/mm²)/(mm³/sec)). In someembodiments, the hydrodynamic resistance between the electrode reservoirand the intersection of the inlet fluid channel and outlet fluid channelis greater than 1 ((N/mm²)/(mm³/sec)). In some embodiments, theelectrical resistance between the electrode reservoir and theintersection of the inlet fluid channel and outlet fluid channel is lessthan 10,000,000 ohms. In some embodiments, the electrical resistancebetween the electrode reservoir and the intersection of the inlet fluidchannel and outlet fluid channel is less than 100,000 ohms. In someembodiments, during operation, the electrode reservoir is filled withthe electrolyte solution at a concentration between about 1 millimolar(mM) to about 500 mM. In some embodiments, during operation, theelectrode reservoir is filled with the electrolyte solution at aconcentration between about 10 mM to about 150 mM. In some embodiments,during operation, the electrode reservoir is filled with the electrolytesolution with a pH range between about 1.5 and about 14. In someembodiments, the separation channel comprises a lumen of a capillary. Insome embodiments, the separation channel comprises a fluid channelwithin a microfluidic device. In some embodiments, the separationchannel is configured to perform electrophoresis. In some embodiments,the separation channel is configured to perform isoelectric focusing.

Also disclosed herein is a fluidic device comprising: at least one fluidinlet; at least one fluid outlet; at least one separation channelcomprising a first end that is fluidically coupled to the at least onefluid inlet and a second end that is fluidically coupled to the at leastone fluid outlet; wherein at least one fluid inlet or at least one fluidoutlet is electrically coupled to a high voltage electrode using afixture comprising: an electrode reservoir; an inlet fluid channelcomprising a first end and a second end; an outlet fluid channelcomprising a first end that is fluidically coupled to the second end ofthe inlet fluid channel, and a second end that is fluidically coupled toone of the at least one fluid inlet or the at least one fluid outlet,wherein the inlet fluid channel and the outlet fluid channel intersectwith and are fluidically coupled to each other at a plane that definesor is parallel to a surface of the electrode reservoir; and a membranedisposed within the electrode reservoir at or adjacent the plane suchthat it covers all or substantially all of an opening comprising theintersection of the inlet fluid channel and the outlet fluid channel;wherein the membrane provides a high hydrodynamic resistance, lowelectrical resistance connection between a high voltage electrodepositioned within the electrode reservoir and a fluid contained withinthe inlet fluid channel and outlet fluid channel.

In some embodiments, the device comprises at least one capillary, andwherein the at least one capillary comprises a lumen which functions asthe at least one separation channel. In some embodiments, the device isa microfluidic device comprising a planar substrate, and wherein theplanar substrate comprises the at least one separation channel. In someembodiments, the at least one fluid inlet or the at least one fluidoutlet is disposed on at least one edge of the planar substrate. In someembodiments, the membrane is hydrophilic. In some embodiments, themembrane comprises a regenerated cellulose membrane. In someembodiments, the membrane comprises a woven polytetrafluoroethylene(PTFE) membrane that has been treated to be hydrophilic. In someembodiments, a cross-sectional area of the membrane or opening isbetween about 0.001 mm² and 100 mm². In some embodiments, the electrodereservoir further comprises an insert disposed within the electrodereservoir and positioned at or adjacent to the membrane, wherein theinsert comprises an inlet fluid path and an outlet fluid path thatfacilitate substantially bubble-free wetting of a surface of themembrane when the electrode reservoir is filled with an electrolytesolution. In some embodiments, during operation, the electrode reservoiris filled with the electrolyte solution at a concentration between about1 millimolar (mM) to about 500 mM. In some embodiments, duringoperation, the electrode reservoir is filled with the electrolytesolution at a concentration between about 10 mM to about 150 mM. In someembodiments, during operation, the electrode reservoir is filled withthe electrolyte solution with a pH range between about 1.5 and about 14.In some embodiments, the hydrodynamic resistance between the electrodereservoir and the intersection of the inlet fluid channel and outletfluid channel is greater than 0.1 ((N/mm²)/(mm³/sec)). In someembodiments, the hydrodynamic resistance between the electrode reservoirand the intersection of the inlet fluid channel and outlet fluid channelis greater than 1 ((N/mm²)/(mm³/sec)). In some embodiments, theelectrical resistance between the electrode reservoir and theintersection of the inlet fluid channel and outlet fluid channel is lessthan 10,000,000 ohms. In some embodiments, the electrical resistancebetween the electrode reservoir and the intersection of the inlet fluidchannel and outlet fluid channel is less than 100,000 ohms.

In some embodiments, the ratio of the hydrodynamic resistance and theelectrical resistance between the electrode reservoir and theintersection of the inlet fluid channel and outlet fluid channel isgreater than about 0.001 ((N/mm²)/(mm³/sec))/Ohm. In some embodiments,the ratio is greater than about 0.01 ((N/mm²)/(mm³/sec))/Ohm. In someembodiments, the ratio is greater than about 0.1((N/mm²)/(mm³/sec))/Ohm. In some embodiments, the ratio is greater thanabout 1 ((N/mm²)/(mm³/sec))/Ohm. In some embodiments, the ratio isgreater than about 10 ((N/mm²)/(mm³/sec))/Ohm. In some embodiments, theratio is greater than about 100 ((N/mm²)/(mm³/sec))/Ohm. In someembodiments, the ratio is greater than about 1000((N/mm²)/(mm³/sec))/Ohm. In some embodiments, the ratio is greater thanabout 10000 ((N/mm²)/(mm³/sec))/Ohm.

In another aspect, disclosed herein are methods for performing aplurality of isoelectric focusing reactions in parallel, the methodcomprising: a) providing a device comprising a planar substrate, whereinthe planar substrate comprises a plurality of separation channels; b)introducing a sample comprising a mixture of analytes into at least twoseparation channels of the plurality of separation channels; c)controlling a voltage applied to the at least two separation channels toperform the plurality of isoelectric focusing reactions to separate themixture of analytes of the sample in the at least two separationchannels; and d) independently monitoring a current flowing through theat least two separation channels as the plurality of isoelectricfocusing reactions are performed in parallel.

In some embodiments, a first end of the at least two separation channelsof the plurality of separation channels is electrically coupled to ananolyte reservoir using a membrane-containing high voltage electrodefixture. In some embodiments, a second end of the at least twoseparation channels of the plurality of separation channels iselectrically coupled to a catholyte reservoir using amembrane-containing high voltage electrode fixture. In some embodiments,voltages applied to the at least two separation channels areindependently controlled. In some embodiments, the samples introducedinto the at least two separation channels are the same, and a first setof experimental conditions is used to perform the isoelectric focusingreactions in a first subset of the at least two separation channels andan at least second set of experimental conditions is used to perform theisoelectric focusing reactions in an at least second subset of the atleast two separation channels. In some embodiments, a different set ofexperimental conditions is used to perform the isoelectric focusingreactions in each of the at least two separation channels. In someembodiments, a set of experimental conditions used to perform theplurality of isoelectric focusing reactions comprises at least onemember of the group consisting of: a buffer selection, a pH gradientselection, a voltage setting, a current setting, an electric fieldstrength setting, a time course for varying a voltage setting, a currentsetting, an electric field strength setting, or an isoelectric focusingreaction time. In some embodiments, the samples introduced into the atleast two separation channels are different for at least two subsets ofthe at least two separation channels and the same set of experimentalconditions are used to perform the isoelectric focusing reactions ineach of the at least two separation channels. In some embodiments, thesamples introduced into each of the at least two separation channels aredifferent. In some embodiments, the method further comprises recording acurrent trace for each of the at least two separation channels whileperforming the plurality of isoelectric focusing reactions. In someembodiments, the method further comprises flushing the at least twoseparation channels following completion of the isoelectric focusingreactions and introducing another sample into the at least twoseparation channels in an automated fashion. In some embodiments,detection of a failure in any of the at least two separation channelstriggers an automated re-introduction and repeat of the isoelectricfocusing reaction for the sample that had been introduced into thatseparation channel. In some embodiments, the failure comprisesintroduction of a bubble, formation of a bubble, an incorrectly preparedsample, an underfilled reagent reservoir, or any combination thereof. Insome embodiments, the failure is detected by monitoring the currentflowing through a separation channel or by processing an image of theseparation channel. In some embodiments, the method further comprisesmeasuring dynamic light scattering in at least one of the at least twoseparation channels while performing isoelectric focusing. In someembodiments, the measurement of dynamic light scattering provides adetermination of a size distribution profile, a determination of anaggregation state, or a determination of a hydrodynamic radius for oneor more separated analytes. In some embodiments, the membrane-containinghigh voltage electrode fixture comprises: a) an electrode reservoir; b)an inlet fluid channel comprising a first end and a second end; c) anoutlet fluid channel comprising a first end that is fluidically coupledto the second end of the inlet fluid channel, and a second end that isfluidically coupled to a separation channel, wherein the inlet fluidchannel and the outlet fluid channel intersect with and are fluidicallycoupled to each other at a plane that defines or is parallel to asurface of the electrode reservoir; and d) a membrane disposed withinthe electrode reservoir at or adjacent to the plane such that themembrane covers all or substantially all of an opening comprising anintersection of the inlet fluid channel and the outlet fluid channel;wherein the membrane provides a high hydrodynamic resistance, lowelectrical resistance connection between a high voltage electrodepositioned within the electrode reservoir and a fluid contained withinthe inlet fluid channel and the outlet fluid channel. In someembodiments, the membrane is hydrophilic and comprises cellulose orpolytetrafluoroethylene (PTFE). In some embodiments, themembrane-containing high voltage electrode fixture further comprises aninsert disposed within the electrode reservoir and positioned at oradjacent to the membrane, wherein the insert comprises an inlet fluidpath and an outlet fluid path that facilitates substantially bubble-freewetting of a surface of the membrane when the electrode reservoir isfilled with an electrolyte solution. In some embodiments, the methodfurther comprises filling the electrode reservoir with the electrolytesolution at a concentration between about 1 millimolar (mM) to about 500mM. In some embodiments, the method further comprises filling theelectrode reservoir with the electrolyte solution at a concentrationbetween about 10 mM to about 150 mM. In some embodiments, theelectrolyte solution has a pH in a range between about 1.5 and about 14.

In another aspect, disclosed herein is a microfluidic device comprisinga planar substrate, wherein the planar substrate comprises: a) aplurality of fluid inlets, wherein all or a portion of the plurality offluid inlets are located on one or more edges of the planar substrate;and b) a plurality of separation channels comprising: i) a first endthat is electrically coupled to an anolyte reservoir using amembrane-containing high voltage electrode fixture; ii) a second endthat is electrically coupled to a catholyte reservoir using amembrane-containing high voltage electrode fixture; and iii) one of thefirst end or the second end of each separation channel of the pluralityof separation channels is in fluid communication with a different fluidinlet of the plurality of fluid inlets.

In some embodiments, the plurality of separation channels is configuredfor UV absorbance imaging or fluorescence imaging of all or a portion ofthe plurality of separation channels. In some embodiments, themicrofluidic device further comprises a cartridge which encompasses allor a portion of a substrate comprising the plurality of separationchannels, and wherein the cartridge comprises a plurality ofmembrane-containing high voltage electrode fixtures. In someembodiments, the cartridge is a disposable component of a systemconfigured to perform multiplexed isoelectric focusing reactions.

Also disclosed herein is a system for performing multiplexed isoelectricfocusing reactions, the system comprising: a) a microfluidic devicecomprising a planar substrate, wherein the planar substrate comprises:i) a plurality of fluid inlets, wherein all or a portion of theplurality of fluid inlets are located on one or more edges of the planarsubstrate; and ii) a plurality of separation channels comprising: afirst end that is electrically coupled to an anolyte reservoir using amembrane-containing high voltage electrode fixture; a second end that iselectrically coupled to a catholyte reservoir using amembrane-containing high voltage electrode fixture; and one of the firstend or the second end of each separation channel of the plurality ofseparation channels is in fluid communication with a different fluidinlet of the plurality of fluid inlets; and b) a multiplexed powersupply, wherein the multiplexed power supply is configured to: i)control a voltage applied to each of at least two separation channels;and ii) independently monitor a current flowing through each of the atleast two separation channels while performing multiplexed isoelectricfocusing reactions for a plurality of samples comprising mixtures ofanalytes.

In some embodiments, the multiplexed power supply is configured toindependently control the voltage applied to each of the at least twoseparation channels. In some embodiments, the system further comprisesan imaging unit configured to (i) acquire UV absorbance or fluorescenceimages of all or a portion of each of the at least two separationchannels, and (ii) process the UV absorbance or fluorescence images todetect a position of one or more isoelectric point (pI) markerscontained within a separation channel during operation so that a pIvalue may be determined for one or more separated analyte peaks in eachof the at least two separation channels. In some embodiments, the systemfurther comprises a dynamic light scattering unit configured to measuredynamic light scattering in at least one separation channel of theplurality of separation channels. In some embodiments, the systemfurther comprises an automated liquid handling system for loadingsamples into sample inlet ports that are fluidically-coupled via theplurality of fluid inlets to the plurality of separation channels. Insome embodiments, the system is configured to flush the plurality ofseparation channels following completion of the multiplexed isoelectricfocusing reactions and introduce another set of samples into theplurality of separation channels in an automated fashion. In someembodiments, detection of a failure in a separation channel triggers anautomated re-introduction and repeat of the isoelectric focusingreaction for the sample that had been introduced into that separationchannel.

Also disclosed herein are methods for performing a plurality ofisoelectric focusing reactions in parallel, the method comprising: a)providing a plurality of sample aliquots, wherein each sample aliquotcomprises a mixture of analytes; b) providing a device comprising aplurality of separation channels, wherein one sample aliquot of theplurality of sample aliquots is introduced into each separation channel;and c) providing a multiplexed power supply, wherein the multiplexedpower supply is configured to independently control and monitor acurrent flowing through each of the plurality of separation channelswhile performing a plurality of isoelectric focusing reactions toseparate the analytes in each sample.

In some embodiments, the sample aliquot introduced into each separationchannel is drawn from the same sample and a different set ofexperimental conditions is used to perform the isoelectric focusingreactions in at least two subsets of the plurality of separationchannels. In some embodiments, a different set of experimentalconditions is used to perform the plurality of isoelectric focusingreactions in each of the plurality of separation channels. In someembodiments, at least two subsets of the plurality of sample aliquotsintroduced into the separation channels are drawn from different samplesand the same set of experimental conditions are used to perform theplurality of isoelectric focusing reactions. In some embodiments, eachof the sample aliquots introduced into the separation channels is drawnfrom a different sample. In some embodiments, the set of experimentalconditions used to perform the plurality of isoelectric focusingreactions comprises a buffer selection, a pH gradient selection, avoltage setting, a current setting, an electric field strength setting,a time course for varying a voltage setting, a current setting, or anelectric field strength setting, an isoelectric focusing reaction time,or any combination thereof. In some embodiments, the analytes compriseproteins. In some embodiments, the device comprises a microfluidicdevice. In some embodiments, the microfluidic device comprises from 4 to8 separation channels. In some embodiments, the sample aliquots areintroduced into the separation channels using an automated liquidhandling system. In some embodiments, the method further comprisesrecording a current trace for each of the plurality of separationchannels while performing the plurality of isoelectric focusingreactions. In some embodiments, the method further comprises monitoringthe plurality of isoelectric focusing reactions using an imagingtechnique to detect a position of one or more separated analyte peaks ineach separation channel. In some embodiments, the imaging techniquecomprises a whole channel imaging technique. In some embodiments, theimaging technique comprises UV absorbance imaging or fluorescenceimaging. In some embodiments, fluorescence imaging comprises nativefluorescence imaging. In some embodiments, the method further comprisesusing the imaging technique to detect a position of one or more pImarkers so that a pI value may be determined for one or more separatedanalyte peaks. In some embodiments, the method further comprisesflushing the separation channels following completion of the pluralityof isoelectric focusing reactions and introducing new sample aliquotsinto the separation channels in an automated fashion. In someembodiments, an automated cycle time for introducing the plurality ofsample aliquots, performing the plurality of isoelectric focusingreactions, and flushing the separation channels is between 1 minute and30 minutes. In some embodiments, detection of an isoelectric focusingreaction failure in any of the plurality of separation channels triggersan automated re-introduction and repeat of the isoelectric focusingreaction for the sample aliquot that had been introduced into thatseparation channel. In some embodiments, the isoelectric focusingreaction failure comprises introduction of a bubble, formation of abubble, or any combination thereof. In some embodiments, the isoelectricfocusing reaction failure is detected by monitoring the current flowingthrough a separation channel or by processing an image of the separationchannel. In some embodiments, the method further comprises measuringdynamic light scattering in at least one channel of the plurality ofseparation channels while performing isoelectric focusing. In someembodiments, the measurement of dynamic light scattering provides adetermination of a size distribution profile for one or more separatedanalytes. In some embodiments, the measurement of dynamic lightscattering provides a determination of an aggregation state for one ormore separated analytes. In some embodiments, the measurement of dynamiclight scattering provides a determination of a hydrodynamic radius forone or more separated analytes.

Also disclosed herein are microfluidic devices comprising: a) aplurality of inlet ports; b) a substrate comprising a plurality ofseparation channels, wherein a proximal end of each separation channelis in fluid communication with a different inlet port; and c) aplurality of outlet ports, wherein a distal end of each separationchannel is in fluid communication with a different outlet port; whereinthe channels of the plurality of separation channels are configured forwhole channel imaging.

In some embodiments, the microfluidic device further comprises anintegrated pair of electrodes for each separation channel, wherein oneelectrode of each pair is in contact with the proximal end of aseparation channel, and the other electrode is in contact with thedistal end of the separation channel. In some embodiments, themicrofluidic device comprises from 4 to 8 separation channels. In someembodiments, the plurality of separation channels is configured forwhole channel UV absorbance imaging. In some embodiments, the pluralityof separation channels is configured for whole channel fluorescenceimaging. In some embodiments, at least one channel within the pluralityof separation channels is configured for performing dynamic lightscattering measurements. In some embodiments, there are no reservoirs orwells on the device. In some embodiments, the inlet ports are located onone or more edges of the device as illustrated in FIG. 1A. In someembodiments, the outlet ports are located on one or more edges of thedevice as illustrated in FIG. 1A. In some embodiments, the microfluidicdevice is a disposable component of a system for performing isoelectricfocusing reactions. In some embodiments, the microfluidic device furthercomprises a cartridge which encompasses all or a portion of thesubstrate, the plurality of inlet ports, or the plurality of outletports. In some embodiments, the cartridge is a disposable component of asystem for performing isoelectric focusing reactions. In someembodiments, one or more inlet ports or outlet ports are interfaced witha high voltage electrode using a membrane-containing high voltageelectrode fixture that provides a bubble-free electrical connection. Insome embodiments, the membrane-containing high voltage electrode fixtureis that illustrated in FIG. 12. In some embodiments, the membranecomprises a hydrophilic membrane. In some embodiments, the membranecomprises a regenerated cellulose membrane. In some embodiments, themembrane comprises a woven polytetrafluoroethylene (PTFE) membrane thathas been treated to be hydrophilic. In some embodiments, amembrane-covered fluid port within the membrane-containing high voltageelectrode fixture has a diameter ranging from about 0.5 to about 2 mm.In some embodiments, an electrode reservoir within themembrane-containing high voltage electrode fixture comprises an insertpositioned within and at the bottom of the electrode reservoir, whereinthe insert comprises an inlet fluid path and an outlet fluid path thatallow bubble-free wetting of a surface of the membrane when theelectrode reservoir is filled. In some embodiments, the cartridgecomprises at least one integrated membrane-containing high voltageelectrode fixture. In some embodiments, the cartridge comprises at leastone reagent reservoir. In some embodiments, the at least one reagentreservoir comprises an anolyte reservoir, a catholyte reservoir, or amobilization reagent reservoir. In some embodiments, the cartridgecomprises at least one flow restrictor. In some embodiments, thecartridge comprises at least one valve. In some embodiments, the atleast one valve comprises a shear valve. In some embodiments, the shearvalve comprises a valve design as illustrated in FIG. 19A. In someembodiments, the shear valve comprises a valve design as illustrated inFIG. 19B. In some embodiments, the cartridge is of a side-manifolddesign as illustrated in any one of FIG. 16, 17, 18, 19A, or 19B so thatclearance is provided for imaging the plurality of separation channels.

Disclosed herein are systems for performing a plurality of isoelectricfocusing reactions in parallel, the system comprising: a) a microfluidicdevice comprising a plurality of separation channels, wherein the devicecomprises: i) a plurality of inlet ports; ii) a substrate comprising aplurality of separation channels, wherein a proximal end of eachseparation channel is in fluid communication with a different inletport; and iii) a plurality of outlet ports, wherein a distal end of eachseparation channel is in fluid communication with a different outletport; wherein the channels of the plurality of separation channels areconfigured for whole channel imaging; and b) a programmable multiplexedpower supply, wherein the multiplexed power supply is configured toindependently control and monitor a current flowing through each of theplurality of separation channels in the microfluidic device whileperforming a plurality of isoelectric focusing reactions to separatemixtures of analytes into their individual components.

In some embodiments, the microfluidic device further comprises anintegrated pair of electrodes for each separation channel, wherein oneelectrode of each pair is in contact with the proximal end of aseparation channel, and the other electrode is in contact with thedistal end of the separation channel. In some embodiments, theseparation channels share a common cathode. In some embodiments, theseparation channels share a common anode. In some embodiments, themicrofluidic device comprises from 4 to 8 separation channels. In someembodiments, the plurality of separation channels is configured forwhole channel UV absorbance imaging. In some embodiments, the pluralityof separation channels is configured for whole channel fluorescenceimaging. In some embodiments, at least one channel within the pluralityof separation channels is configured for performing dynamic lightscattering measurements. In some embodiments, there are no reservoirs orwells on the microfluidic device. In some embodiments, the inlet portsare located on one or more edges of the microfluidic device asillustrated in FIG. 1A. In some embodiments, the outlet ports arelocated on one or more edges of the device as illustrated in FIG. 1A. Insome embodiments, the microfluidic device is a disposable component ofthe system. In some embodiments, the microfluidic device furthercomprises a cartridge which encompasses all or a portion of thesubstrate, the plurality of inlet ports, or the plurality of outletports. In some embodiments, the cartridge is a disposable component ofthe system. In some embodiments, one or more inlet ports or outlet portsare interfaced with a high voltage electrode using a membrane-containinghigh voltage electrode fixture that provides a bubble-free electricalconnection. In some embodiments, the membrane-containing high voltageelectrode fixture is that illustrated in FIG. 12. In some embodiments,the membrane comprises a hydrophilic membrane. In some embodiments, themembrane comprises a regenerated cellulose membrane. In someembodiments, the membrane comprises a woven polytetrafluoroethylene(PTFE) membrane that has been treated to be hydrophilic. In someembodiments, a membrane-covered fluid port within themembrane-containing high voltage electrode fixture has a diameterranging from about 0.5 to about 2 mm. In some embodiments, an electrodereservoir within the membrane-containing high voltage electrode fixturecomprises an insert positioned within and at the bottom of the electrodereservoir, wherein the insert comprises an inlet fluid path and anoutlet fluid path that allow bubble-free wetting of a surface of themembrane when the electrode reservoir is filled. In some embodiments,the cartridge comprises at least one integrated membrane-containing highvoltage electrode fixture. In some embodiments, the cartridge comprisesat least one reagent reservoir. In some embodiments, the at least onereagent reservoir comprises an anolyte reservoir, a catholyte reservoir,or a mobilization reagent reservoir. In some embodiments, the cartridgecomprises at least one flow restrictor. In some embodiments, thecartridge comprises at least one valve. In some embodiments, the atleast one valve comprises a shear valve. In some embodiments, the shearvalve comprises a valve design as illustrated in FIG. 19A. In someembodiments, the shear valve comprises a valve design as illustrated inFIG. 19B. In some embodiments, the cartridge is of a side-manifolddesign as illustrated in any one of FIG. 16, 17, 18, 19A, or 19B so thatclearance is provided for imaging the plurality of separation channels.In some embodiments, the system further comprises an automated liquidhandling system for loading a sample aliquot into each inlet port. Insome embodiments, the automated liquid handling system is configured forintroduction of sample aliquots into each separation channel that areall drawn from the same sample, and the system is configured to use adifferent set of experimental conditions to perform the isoelectricfocusing reactions in at least two subsets of the plurality ofseparation channels. In some embodiments, a different set ofexperimental conditions is used to perform the isoelectric focusingreactions in each of the plurality of separation channels. In someembodiments, the automated liquid handling system is configured forintroduction of at least two subsets of sample aliquots into theseparation channels that are drawn from different samples and the sameset of experimental conditions are used to perform the plurality ofisoelectric focusing reactions. In some embodiments, each of the samplealiquots introduced into the separation channels is drawn from adifferent sample. In some embodiments, the set of experimentalconditions used to perform the plurality of isoelectric focusingreactions comprises a buffer selection, a pH gradient selection, avoltage setting, a current setting, an electric field strength setting,a time course for varying a voltage setting, a current setting, or anelectric field strength setting, an isoelectric focusing reaction time,or any combination thereof. In some embodiments, the analytes compriseproteins. In some embodiments, the programmable multiplexed power supplyis further configured to record a current trace for each of theplurality of separation channels while the isoelectric focusingreactions are being performed. In some embodiments, the system furthercomprises an imaging unit configured to acquire images of, and detectseparated analyte peaks in, the plurality of separation channels. Insome embodiments, the imaging unit is configured to perform wholechannel imaging of the plurality of separation channels. In someembodiments, the imaging unit is configured to acquire UV absorbanceimages. In some embodiments, the imaging unit is configured to acquirefluorescence images. In some embodiments, the fluorescence imagescomprise native fluorescence images. In some embodiments, the imagingunit is further configured to detect a position of one or more pImarkers so that a pI value may be determined for one or more separatedanalyte peaks. In some embodiments, the system is configured to flushthe separation channels following completion of the plurality ofisoelectric focusing reactions and introduce a new sample aliquot intothe separation channels in an automated fashion. In some embodiments, anautomated cycle time for introducing the plurality of sample aliquots,performing the plurality of isoelectric focusing reactions, and flushingthe separation channels is between 1 minute and 30 minutes. In someembodiments, detection of an isoelectric focusing reaction failure inany of the plurality of separation channels triggers an automatedre-introduction and repeat of the isoelectric focusing reaction for thesample aliquot that had been introduced into that separation channel. Insome embodiments, the isoelectric focusing reaction failure comprisesintroduction of a bubble, formation of a bubble, or any combinationthereof. In some embodiments, the isoelectric focusing reaction failurecomprises an improperly prepared sample. In some embodiments, theisoelectric focusing reaction failure comprises an empty or underfilledsample well. In some embodiments, the isoelectric focusing reactionfailure is detected by monitoring the current flowing through aseparation channel or by processing an image of the separation channel.In some embodiments, the system further comprises a dynamic lightscattering measurement unit. In some embodiments, the system furthercomprises a fluid flow controller configured to provide independentlycontrolled pressure-driven flow through one or more separation channels,one or more mobilizer channels, or one or more auxiliary fluid channels.In some embodiments, the pressure-driven flow through the one or moreseparation channels, one or more mobilizer channels, or one or moreauxiliary fluid channels is pulse-less flow. In some embodiments, thesystem further comprises a temperature controller configured to maintainthe plurality of separation channels at a constant temperature.

Also disclosed herein are methods, comprising: a) applying an electricfield across a separation channel in a microfluidic device to perform aseparation of an analyte mixture via isoelectric focusing; b)simultaneously and continuously imaging the separation and mobilizationof the separated analyte mixture in the whole separation channel or aportion thereof; and c) expelling separated and mobilized analytes viaelectrospray ionization from an orifice on the microfluidic device intoa mass spectrometer; wherein the orientation of the microfluidic deviceis tilted relative to a horizontal plane such that the orifice isdirected downwards towards an inlet of the mass spectrometer. In someembodiments, the method further comprises correlating separated analytepeaks detected in the separation channel with mass spectrometer data forthe separated analytes. In some embodiments, the separated analyte peaksare detected by absorbance imaging. In some embodiments, the separatedanalyte peaks are detected by fluorescence imaging. In some embodiments,the orifice is in electrical communication with the separation channel'selectric field. In some embodiments, the orifice is a recess on themicrofluidic device, such that a Taylor cone formed by electrosprayionization is disposed entirely within the recess. In some embodiments,the microfluidic device comprises a first separation channel and asecond separation channel. In some embodiments, the method furthercomprises: chromatographically-enriching the analyte mixture in thefirst separation channel before applying the electric field to performthe isoelectric focusing separation of the analyte mixture in the secondseparation channel. In some embodiments, the method further comprisesintroducing ampholytes into the separation channel before the separationof the analyte mixture to generate a pH gradient in the separationchannel, introducing isoelectric point (pI) markers into the separationchannel before the separation, and continuously imaging the separationchannel while the pI markers are separated. In some embodiments, theanalyte mixture comprises intact proteins. In some embodiments, themobilization is performed by introducing an electrolyte into theseparation channel using pressure. In some embodiments, the mobilizationis performed by introducing an electrolyte into the separation channelby electrophoresis. In some embodiments, the mobilization is performedby introducing an electrolyte into the separation channel from anelectrolyte channel in fluid communication with a confluence regiondownstream of the separation channel. In some embodiments, themicrofluidic device comprises two electrodes to generate an electricfield across an electrolyte introducing channel. In some embodiments,there are no reservoirs or wells on the microfluidic device. In someembodiments, inlet ports for the microfluidic device are located on oneor more edges of the device as illustrated in FIG. 2. In someembodiments, outlet ports for the microfluidic device are located on oneor more edges of the device as illustrated in FIG. 2. In someembodiments, the microfluidic device is a disposable component of asystem for performing isoelectric focusing and mass spectrometricanalysis. In some embodiments, the microfluidic device is a cartridgecomprising the separation channel, the orifice, the electrolyteintroducing channel, an anolyte introducing channel, and gas deliverychannels for ionization. In some embodiments, the cartridge is adisposable component of a system for performing isoelectric focusing andmass spectrometric analysis. In some embodiments, one or more inletports or outlet ports of the microfluidic device are interfaced with ahigh voltage electrode using a membrane-containing high voltageelectrode fixture that provides a bubble-free electrical connection. Insome embodiments, the membrane-containing high voltage electrode fixtureis that illustrated in FIG. 12. In some embodiments, the membranecomprises a hydrophilic membrane. In some embodiments, the membranecomprises a regenerated cellulose membrane. In some embodiments, themembrane comprises a woven polytetrafluoroethylene (PTFE) membrane thathas been treated to be hydrophilic. In some embodiments, amembrane-covered fluid port within the membrane-containing high voltageelectrode fixture has a diameter ranging from about 0.5 to about 2 mm.In some embodiments, an electrode reservoir within themembrane-containing high voltage electrode fixture comprises an insertpositioned within and at the bottom of the electrode reservoir, whereinthe insert comprises an inlet fluid path and an outlet fluid path thatallow bubble-free wetting of a surface of the membrane when theelectrode reservoir is filled. In some embodiments, the cartridgecomprises at least one integrated membrane-containing high voltageelectrode fixture. In some embodiments, the cartridge comprises at leastone reagent reservoir. In some embodiments, the at least one reagentreservoir comprises an anolyte reservoir, a catholyte reservoir, or amobilization reagent reservoir. In some embodiments, the cartridgecomprises at least one flow restrictor. In some embodiments, thecartridge comprises at least one valve. In some embodiments, the atleast one valve comprises a shear valve. In some embodiments, the shearvalve comprises a valve design as illustrated in FIG. 19A. In someembodiments, the shear valve comprises a valve design as illustrated inFIG. 19B. In some embodiments, the cartridge is of a side-manifolddesign as illustrated in any one of FIG. 16, 17, 18, 19A, or 19B so thatclearance is provided for imaging the plurality of separation channels.In some embodiments, the method further comprises providing an automatedliquid handling system for loading a sample aliquot into each inletport.

Disclosed herein are microfluidic devices comprising a substrate,wherein the substrate defines: a) one or more inlet ports; b) at leastone separation channel configured to perform separation of an analytemixture; and c) an orifice in fluid communication with an end of theseparation channel, wherein the orifice is configured to performelectrospray ionization of separated analyte fractions eluted from theseparation channel and expel them into a mass spectrometer.

In some embodiments, the microfluidic device further comprises anintegrated pair of electrodes for the at least one separation channel,wherein one electrode of each pair is in contact with a proximal end ofa separation channel, and the other electrode is in contact with adistal end of the separation channel. In some embodiments, the at leastone separation channel is configured for whole channel UV absorbanceimaging. In some embodiments, the at least one separation channel isconfigured for whole channel fluorescence imaging. In some embodiments,there are no reservoirs or wells on the device. In some embodiments, theone or more inlet ports are located on one or more edges of the deviceas illustrated in FIG. 2. In some embodiments, the device furthercomprises an auxiliary fluid channel used to deliver a calibrantsolution for calibrating mass data. In some embodiments, themicrofluidic device is a disposable component of a system for performingisoelectric focusing and mass spectrometric analysis of analytemixtures. In some embodiments, the microfluidic device further comprisesa cartridge which encompasses all or a portion of the substrate, the oneor more inlet ports, or the orifice. In some embodiments, the cartridgeis a disposable component of a system for performing isoelectricfocusing and mass spectrometric analysis of analyte mixtures. In someembodiments, one or more inlet ports are interfaced with a high voltageelectrode using a membrane-containing high voltage electrode fixturethat provides a bubble-free electrical connection. In some embodiments,the membrane-containing high voltage electrode fixture is thatillustrated in FIG. 12. In some embodiments, the membrane comprises ahydrophilic membrane. In some embodiments, the membrane comprises aregenerated cellulose membrane. In some embodiments, the membranecomprises a woven polytetrafluoroethylene (PTFE) membrane that has beentreated to be hydrophilic. In some embodiments, the membrane comprises arigid material, e.g., glass or ceramic. In some embodiments, themembrane may be treated to be hydrophilic and/or uncharged. In someembodiments, a membrane-covered fluid port within themembrane-containing high voltage electrode fixture has a diameterranging from about 0.5 to about 2 mm. In some embodiments, an electrodereservoir within the membrane-containing high voltage electrode fixturecomprises an insert positioned within and at the bottom of the electrodereservoir, wherein the insert comprises an inlet fluid path and anoutlet fluid path that allow bubble-free wetting of a surface of themembrane when the electrode reservoir is filled. In some embodiments,the cartridge comprises at least one integrated membrane-containing highvoltage electrode fixture. In some embodiments, the cartridge comprisesat least one reagent reservoir. In some embodiments, the at least onereagent reservoir comprises an anolyte reservoir, a catholyte reservoir,or a mobilization reagent reservoir. In some embodiments, the cartridgecomprises at least one flow restrictor. In some embodiments, thecartridge comprises at least one valve. In some embodiments, the atleast one valve comprises a shear valve. In some embodiments, the shearvalve comprises a valve design as illustrated in FIG. 19A. In someembodiments, the shear valve comprises a valve design as illustrated inFIG. 19B. In some embodiments, the cartridge comprises a mechanism tofacilitate application of a vacuum to remove excess fluid build-up froman exterior surface of the orifice. In some embodiments, the cartridgecomprises a nebulizer mechanism to facilitate formation of a stableTaylor cone. In some embodiments, the cartridge is of a side-manifolddesign as illustrated in any one of FIG. 16, 17, 18, 19A, or 19B so thatclearance is provided for imaging the plurality of separation channels.

Disclosed herein are apparatus comprising: a) a microfluidic devicecomprising a substrate, wherein the substrate defines: i) one or moreinlet ports; ii) a separation channel configured to perform separationof an analyte mixture; and iii) an orifice in fluid communication withan end of the separation channel, wherein the orifice is configured toperform electrospray ionization of separated analyte fractions elutedfrom the separation channel and expel them into a mass spectrometer,wherein the orientation of the microfluidic device is tilted relative toa horizontal plane such that the orifice is directed downwards towardsan inlet of the mass spectrometer; and b) an imaging device configuredto simultaneously and continuously image the separation and elution ofthe separated analyte fractions in the entire separation channel or aportion thereof.

In some embodiments, the system further comprises a mass spectrometer.In some embodiments, the apparatus is further configured to correlateseparated analyte peaks detected in the separation channel with massspectrometer data for the separated analytes. In some embodiments, theseparation comprises a chromatographic separation. In some embodiments,the apparatus further comprises at least two electrodes, wherein the atleast two electrodes are configured to apply an electric field acrossthe separation channel to separate the analyte mixture via isoelectricfocusing. In some embodiments, the apparatus further comprises at leasttwo electrodes, wherein the at least two electrodes are configured toapply an electric field across the separation channel to separate theanalyte mixture via electrophoresis. In some embodiments, peakscorresponding to the separated analyte fractions are detected byabsorbance imaging. In some embodiments, peaks corresponding to theseparated analyte fractions are detected by fluorescence imaging. Insome embodiments, the orifice is in electrical communication with theseparation channel's electric field. In some embodiments, the orifice ispositioned in a recess on the microfluidic device, such that a Taylorcone formed by electrospray ionization is disposed entirely within therecess. In some embodiments, an external surface of the orificecomprises a hydrophobic coating to prevent excess fluid build-up duringoperation. In some embodiments, the microfluidic device comprises afirst separation channel and a second separation channel. In someembodiments, the microfluidic device is configured to perform achromatographic-enrichment of the analyte mixture in the firstseparation channel before applying the electric field to performisoelectric focusing or electrophoretic separation of the analytemixture in the second separation channel. In some embodiments,isoelectric point (pI) markers are introduced into the separationchannel prior to performing an isoelectric focusing separation and areused to map pI ranges in the separation channel. In some embodiments,the apparatus is further configured to determine a value for theisoelectric point of at least one separated analyte fraction. In someembodiments, the analyte mixture comprises intact proteins. In someembodiments, mobilization is performed by introducing an electrolyteinto the separation channel using pressure. In some embodiments,mobilization is performed by introducing an electrolyte into theseparation channel using electrophoresis. In some embodiments, themicrofluidic device further comprises an additional electrolyteintroducing channel that intersects the separation channel at theconfluence of the separation channel and orifice, and wherein themobilization is performed by introducing an electrolyte into theseparation channel from the electrolyte introducing channel. In someembodiments, the apparatus further comprises two electrodes configuredto apply an electric field across the electrolyte introducing channel tointroduce a mobilization electrolyte. In some embodiments, themicrofluidic device further comprises an anolyte introducing channel andgas delivery channels for ionization. In some embodiments, themicrofluidic device further comprises an optical slit aligned with theseparation channel such that light is only transmitted through theoptical slit. In some embodiments, there are no reservoirs or wells onthe microfluidic device. In some embodiments, the one or more inletports of the microfluidic device are located on one or more edges of thedevice as illustrated in FIG. 2. In some embodiments, the microfluidicdevice is a disposable component of the apparatus. In some embodiments,the microfluidic device further comprises a cartridge which encompassesall or a portion of the substrate, the one or more inlet ports, and theorifice. In some embodiments, the cartridge is a disposable component ofthe apparatus. In some embodiments, one or more inlet ports areinterfaced with a high voltage electrode using a membrane-containinghigh voltage electrode fixture that provides a bubble-free electricalconnection. In some embodiments, the membrane-containing high voltageelectrode fixture is that illustrated in FIG. 12. In some embodiments,the membrane comprises a hydrophilic membrane. In some embodiments, themembrane comprises a regenerated cellulose membrane. In someembodiments, the membrane comprises a woven polytetrafluoroethylene(PTFE) membrane that has been treated to be hydrophilic. In someembodiments, a membrane-covered fluid port within themembrane-containing high voltage electrode fixture has a diameterranging from about 0.5 to about 2 mm. In some embodiments, an electrodereservoir within the membrane-containing high voltage electrode fixturecomprises an insert positioned within and at the bottom of the electrodereservoir, wherein the insert comprises an inlet fluid path and anoutlet fluid path that allow bubble-free wetting of a surface of themembrane when the electrode reservoir is filled. In some embodiments,the cartridge comprises at least one integrated membrane-containing highvoltage electrode fixture. In some embodiments, the cartridge comprisesat least one reagent reservoir. In some embodiments, the at least onereagent reservoir comprises an anolyte reservoir, a catholyte reservoir,or a mobilization reagent reservoir. In some embodiments, the cartridgecomprises at least one flow restrictor. In some embodiments, thecartridge comprises at least one valve. In some embodiments, the atleast one valve comprises a shear valve. In some embodiments, the shearvalve comprises a valve design as illustrated in FIG. 19A. In someembodiments, the shear valve comprises a valve design as illustrated inFIG. 19B. In some embodiments, the cartridge comprises a mechanism tofacilitate application of a vacuum to remove excess fluid build-up froman exterior surface of the orifice. In some embodiments, the apparatusfurther comprises a mechanism to facilitate application of a vacuum toremove excess fluid build-up from an exterior surface of the orifice. Insome embodiments, the apparatus further comprises a wiper mechanism toremove excess fluid build-up from an exterior surface of the orifice. Insome embodiments, the cartridge comprises a nebulizer mechanism tofacilitate formation of a stable Taylor cone. In some embodiments, theapparatus further comprises a nebulizer mechanism to facilitateformation of a stable Taylor cone. In some embodiments, the cartridge isof a side-manifold design as illustrated in any one of FIG. 16, 17, 18,19A, or 19B so that clearance is provided for imaging the plurality ofseparation channels. In some embodiments, the apparatus furthercomprises a fluid flow controller configured to provide independentlycontrolled pressure-driven flow through one or more separation channels,one or more mobilizer channels, or one or more auxiliary fluid channels.In some embodiments, the pressure-driven flow through the one or moreseparation channels, one or more mobilizer channels, or one or moreauxiliary fluid channels is pulse-less flow. In some embodiments, theapparatus further comprises a temperature controller configured tomaintain the plurality of separation channels at a constant temperature.In some embodiments, the microfluidic device comprises an auxiliaryfluid channel used to deliver a calibrant solution for calibrating massdata. In some embodiments, the imaging device may be configured tofunction as a point detector during elution of analyte fractions fromthe separation channel to provide improved time resolution fortime-based chromatograms. In some embodiments, between 4 and 16 imagingdevice pixels are binned to function as a point detector. In someembodiments, intensity data from the binned pixels is read-out at a rateof at least 1 Hz.

Disclosed herein are fixtures comprising: a) an electrode reservoir; b)an inlet fluid channel and an outlet fluid channel that intersect at aplane; and c) a membrane positioned within the electrode reservoir onsaid plane such that it covers an opening comprising the intersection ofthe inlet fluid channel and the outlet fluid channel; wherein themembrane facilitates the formation of a bubble-free electricalconnection between a high voltage electrode and the fluid within theinlet and outlet fluid channels.

In some embodiments, the fixture comprises a design as illustrated inFIG. 12. In some embodiments, the membrane comprises a hydrophilicmembrane. In some embodiments, the membrane comprises a regeneratedcellulose membrane. In some embodiments, the membrane comprises a wovenpolytetrafluoroethylene (PTFE) membrane that has been treated to behydrophilic. In some embodiments, the opening covered by the membranehas a diameter ranging from about 0.5 to about 2 mm. In someembodiments, the electrode reservoir further comprises an insertpositioned within and at the bottom of the electrode reservoir and ontop of the membrane, wherein the insert comprises an inlet fluid pathand an outlet fluid path that allow bubble-free wetting of a surface ofthe membrane when the electrode reservoir is filled.

Disclosed herein are shear valves comprising a design as illustrated inFIG. 19A or FIG. 19B.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety. In the event of a conflictbetween a term herein and a term in an incorporated reference, the termherein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A provides a non-limiting schematic illustration of a microfluidicdevice comprising a four-channel isoelectric focusing design accordingto one aspect of the present disclosure.

FIG. 1B provides a non-limiting schematic illustration of a fluidchannel network of an exemplary microfluidic device comprising anelectrospray tip of the present disclosure.

FIG. 2 provides a schematic top-down view of one non-limiting example ofa microfluidic device for performing one or more separation reactions,e.g., isoelectric focusing reactions, followed by electrosprayionization.

FIG. 3 provides a cross-section view of one non-limiting example of amicrofluidic device for performing one or more separation reactions,e.g., isoelectric focusing reaction, followed by electrosprayionization.

FIG. 4 provides a schematic of an example waste management system.

FIG. 5 provides a schematic of another example waste management system.

FIG. 6 schematically shows another example of a waste management systemcomprising a clamp module.

FIG. 7 schematically shows another example of a waste management systemcomprising a tube vacuum apparatus.

FIG. 8 schematically shows another example of a waste management systemusing positive pressure.

FIG. 9 schematically illustrates an example nebulizer.

FIGS. 10A-D schematically illustrate additional non-limiting examples ofa nebulizer. FIG. 10A: front view of a second nebulizer design. FIG.10B: isometric view of a second nebulizer design. FIG. 10C: top cut-awayview of a third nebulizer design. FIG. 10D: side cut-away view of athird nebulizer design.

FIGS. 11A-D schematically illustrate a fourth example of a nebulizerdesign. FIG. 11A: partial cut-away view as mounted on microfluidicdevice cartridge. FIG. 11B: stand-alone partial cut-away view. FIG. 11C:cut-away side view. FIG. 11D: cut-away top view.

FIG. 12 schematically illustrates an example of a fixture.

FIGS. 13A-F schematically illustrate a non-limiting example of a fixturecomprising a membrane. FIG. 13A partial cut-away view. FIG. 13B: topview of electrode reservoir. FIG. 13C: partial cut-away view ofelectrode reservoir. FIG. 13D: top view of bottom of electrodereservoir.

FIG. 13E: cut-away detail of bottom of electrode reservoir. FIG. 13F:cut-away side view of bottom of electrode reservoir.

FIG. 14 schematically illustrates an example method of providingreagents to one or more reservoirs of a system.

FIG. 15 illustrates schematically an example method of providingreagents to one or more reservoirs.

FIG. 16 schematically illustrates an example system comprising acartridge, as described in certain embodiments herein.

FIG. 17 shows an example embodiment of a cartridge comprising amicrofluidic device of the present disclosure.

FIG. 18 shows another example embodiment of a cartridge comprising amicrofluidic device of the present disclosure.

FIGS. 19A-B show additional example embodiments of a cartridgecomprising a microfluidic device of the present disclosure and a shearvalve. FIG. 19A: cartridge comprising a rotating shear valve. FIG. 19B:cartridge comprising a spring-loaded shear valve.

FIG. 20 shows another example embodiment of a cartridge comprising amicrofluidic device of the present disclosure.

FIG. 21 schematically shows a non-limiting example of securing featuresthat may be used to secure a device to a cartridge.

FIG. 22 schematically shows a non-limiting example of securing featuresthat may be used to generate a fluidic seal of a cartridge to a device.

FIG. 23 schematically shows a non-limiting example of an electricalconnection to a reservoir of a device or a cartridge.

FIG. 24 schematically shows a non-limiting example of a mounting platefor coupling one or more reservoir units to a cartridge that alsointerface with an instrument.

FIG. 25 shows an example cartridge with a reservoir structure.

FIG. 26 shows another example cartridge with a reservoir structure.

FIG. 27 shows another example cartridge with a reservoir structure.

FIGS. 28 A-D show different perspective views of an example imagingsystem disclosed herein. FIG. 28A: imaging system comprising a scanning(or turning) mirror. FIG. 28B: imaging system comprising a mirror forwhole channel imaging. FIG. 28C: detail view of the imaging system ofFIG. 28B. FIG. 28D: top view of the imaging system illustrated in FIGS.28B and 28C.

FIG. 29 schematically illustrates an example system for interfacing amicrofluidic separation device/cartridge according to an embodimentdescribed herein with a mass spectrometer.

FIG. 30 schematically illustrates another example system, according toan embodiment described herein.

FIG. 31 schematically illustrates an example system, according to anembodiment described herein.

FIGS. 32A-B show example data of a mobilization reaction and a mobilitychromatogram.

FIG. 32A: plot of UV absorbance versus pixel number of the image sensorused to image a separation channel. FIG. 32B: the mobilizationchromatogram (plot of UV absorbance versus time) derived from data suchas that illustrated in FIG. 32A.

FIG. 33 shows a non-limiting example of a software architecture for oneembodiment of the disclosed systems.

FIG. 34 shows a non-limiting example of a block diagram of an integratedsystem in one embodiment of the present disclosure.

FIG. 35 shows another non-limiting example of a block diagram of anintegrated system in one embodiment of the present disclosure.

FIG. 36 provides an exemplary flowchart of a computer-controlled voltagefeedback loop where the ESI tip is held at 0V.

FIG. 37 provides an example flowchart of voltage feedback loop where theESI tip is held at +3000V.

FIG. 38 provides a schematic drawing of an example chip withintersecting channels.

DETAILED DESCRIPTION

Disclosed herein are methods, devices, and systems for performing aplurality of isoelectric focusing reactions (or other separationreactions) in parallel for fast, accurate separation andcharacterization of protein analyte mixtures or other biologicalmolecules by isoelectric point (or other physicochemical properties).

In one aspect of the present disclosure, microfluidic devices comprisingtwo or more separation channels for performing two or more separationreactions in parallel are described, where the microfluidic formatenables fast, accurate separation and characterization of analytemixtures using extremely small input sample volumes. In someembodiments, the microfluidic device comprises a planar substrate, whichplanar substrate comprises the two or more separation channels. In apreferred aspect, the separation reactions are isoelectric focusingreactions. In another preferred aspect, the analyte mixtures compriseprotein analyte mixtures, and the performance of two or more isoelectricfocusing reactions in parallel enables fast, accurate separation of theprotein components in the analyte mixture and characterization of theindividual protein components according to their isoelectric points(pIs). In some instances, the use of imaging, e.g., whole channelimaging, in combination with pI markers to visualize the positions ofthe pI markers in the pH gradient used for isoelectric focusing allowsfor more accurate determinations of the pIs for the separated proteincomponents of the analyte mixture.

In another aspect of the present disclosure, methods and systems foroperating said microfluidic devices are described, where the use of twoor more high voltage power supplies (or a single multiplexed highvoltage power supply), enables independent control of the separationreaction or experimental conditions in each separation channel of themicrofluidic device. Thus, in some instances, the microfluidic devicemay be used to perform separation and characterization of two or moredifferent samples under the same set of separation or experimentalconditions in parallel. In some instances, the microfluidic device maybe used to perform separation and characterization of two or morealiquots of the same sample under two or more different reaction orexperimental conditions in parallel. In some instances, a subset of theseparation channels on the device may be used to perform separations ofa plurality of samples under the same set of separation or experimentalconditions, and, alternatively or in addition to, a different subset ofthe separation channels on the device may be used to perform separationand characterization of a plurality of aliquots from the same sampleunder a plurality of different reaction or experimental conditions inparallel.

The experimental conditions may be the same or may differ across theseparation channels of the microfluidic device and may comprise a bufferselection, an electrolyte selection, a pH gradient selection, a voltagesetting, a current setting, an electric field strength setting, a timecourse for varying a voltage setting, a current setting, an electricfield strength setting, an isoelectric focusing reaction, or acombination thereof.

In some instances, the systems of the present disclosure comprise one ormore of the disclosed microfluidic devices, and two or more high voltagepower supplies (or a single, multiplexed high voltage power supply thatallows independent control of two or more channels). In some instances,the two or more high voltage power supplies (or single, multiplexed highvoltage power supply that allows independent control of two or morechannels) are configured to monitor and/or record the current flowingthrough each separation channel. The monitoring of a separation channel(e.g., the current of the separation channel) may, in some instances, beperformed independently of the monitoring of the other separationchannels. In some instances, the current flowing through each separationchannel may be used, for example, to determine when an isoelectricfocusing reaction is complete and/or to detect a failure (e.g.,introduction or formation of a bubble in a separation channel, anincorrectly prepared sample, an underfilled reagent reservoir, or acombination thereof). In some instances, the failure may be detected bymonitoring the current flowing through a separation channel or byprocessing an image of the separation channel. In some instances, thevoltage supply may be configured to shut off the voltage applied to aseparation channel following detection of a failure. In some instances,the voltage supply may be configured to restart a separation reactionfollowing the detection of a failure.

In some instances, the system may further comprise an autosampler orfluid handling system configured for automated, independently controlledloading of sample aliquots and/or other separation reaction reagentsinto a plurality of sample or reagent inlet ports. In some instances,the system may further comprise a fluid flow controller configured toprovide, e.g., independently controlled pressure-driven flow through twoor more separation channels (e.g., for use alone or in combination witha voltage gradient applied to the two or more separation channels). Insome instances, the system may further comprise an autosampler or fluidflow controller configured to flush, wash, rinse, or evacuate the two ormore separation channels following a separation reaction (e.g.,isoelectric focusing reaction). In some instances, following the flush,wash, rinse, or evacuation of the two or more separation channels, theautosampler or fluid flow controller may be configured to automaticallyintroduce another sample (e.g., a different sample or another aliquot ofthe same sample) into the two or more separation channels. In someinstances, the autosampler or fluid flow controller may be configured toautomatically re-introduce a sample, reaction reagents, or a combinationthereof into the one or more separation channels if a failure (e.g.,bubble formation or introduction, incorrectly prepared sample,underfilled reagent reservoir, or a combination thereof) is detected(e.g., via the voltage or current monitoring). In such cases, followingthe detection of the failure, the autosampler or fluid flow controllermay flush out the separation channel where the failure occurred,re-introduce a sample, reaction reagents, or a combination thereof, andthe separation reaction may be re-initiated (e.g., via application of anelectric field by one or more of the independently controlled voltagesupplies).

In some instances, the system may further comprise an imaging moduleconfigured to acquire a series of one or more images of the two or moreseparation channels. In some instances, the field-of-view of the imagesmay comprise all or a portion of the two or more separation channels. Insome instances, the imaging may comprise continuous imaging while theseparation reactions are performed. In some instances, the imaging maycomprise intermittent imaging while the separation reactions areperformed. In some instances, the imaging may comprise acquiring UVabsorbance images. In some instances, the imaging may comprisefluorescence images, e.g., of either native fluorescence or fluorescencedue to the presence of exogenous fluorescent labels attached to theanalytes. In some instances, the imaging module may be configured, forexample, to determine when an isoelectric focusing reaction is completeand/or to detect a failure (e.g., introduction or formation of a bubblein a separation channel).

In some instances, the system may further comprise a microplate-handlingrobotics module configured to transport and replace microplates thatserve as sources for samples and/or reagents. In some instances, thesystem may further comprise a microfluidic device-handling roboticsmodule configured to transport and replace the microfluidic devices usedin the system, e.g., after a failure is detected. In some instances, themicroplate-handling and the microfluidic device-handling may be handledby the same robotics module.

In another aspect of the present disclosure, systems are described thatmay comprise a microfluidic device designed to perform one or moreseparation reactions, e.g., isoelectric focusing reactions, to separatea sample comprising a mixture of analytes into its individualcomponents, followed by electrospray ionization of the separatedanalytes. In some instances, the microfluidic device may be housed in acartridge that further comprises, e.g., high-voltage electrodeconnections, reagent reservoirs, valves, etc. In some instances, themicrofluidic device may comprise a substantially planar substrate, wherethe planar substrate comprises a plurality of separation channels. Insome instances, a first end of one or more separation channels of theplurality of separation channels is electrically and/or fluidicallycoupled to an electrode (e.g., anolyte) reservoir using a fixture, whichfixture may comprise a membrane. In some instances, a second end of oneor more separation channels is electrically and/or fluidically coupledto an electrode (e.g., catholyte reservoir) using a fixture, whichfixture may comprise a membrane. The membrane may be disposed within theelectrode reservoir at or adjacent to a plane that defines or isparallel to a surface of the electrode reservoir, which plane mayintersect an inlet fluid channel and outlet fluid channel. The membranemay cover all or substantially all of an opening comprising anintersection of the inlet fluid channel and outlet fluid channel. Insome instances, the system may further comprise an analytical instrumentsuch as a mass spectrometer. The disclosed methods, devices, and systemsenable improvements in the reproducibility and quantitative accuracy ofthe separation data, and also improved correlation between theseparation data and downstream analytical characterization data, e.g.,that obtained using a mass spectrometer or other analytical instrument.

A key feature of the disclosed methods, devices, and systems, asindicated above, is the use of imaging to monitor separation reactionsin a separation channel for the purpose of detecting the presence ofanalyte peaks and/or to determine when the separation reaction hasreached completion. In some instances, images may be acquired for all ora portion of the separation channel. In some instances, imaging of allor a portion of the separation channel may be performed while theseparation step and/or a mobilization step are performed. In someinstances, the images may be used to detect the position of enrichedanalyte peaks within the separation channel. In some instances, theimages may be used to detect the presence of one or more markers orindicators, e.g., isoelectric point (pI) standards, within theseparation channel and thus determine the pIs for one or more analytes.In some instances, the images may be used to detect a failure in aseparation channel (e.g. bubble formation). In some instances, dataderived from such images may be used to determine when a separationreaction is complete (e.g., by monitoring peak velocities, peakpositions, and/or peak widths) and subsequently trigger a mobilizationstep.

In some instances, the mobilization step may comprise introduction of amobilization buffer or a mobilization electrolyte into the separationchannel. In some instances, the mobilization buffer or mobilizationelectrolyte may be introduced using hydrodynamic pressure. In someinstances, the mobilization buffer or mobilization electrolyte may beintroduced by means of electrophoresis. In some instances, themobilization buffer or mobilization electrolyte may be introduced bymeans of a combination of electrophoresis and hydrodynamic pressure. Insome instances, the mobilization of a series of one or more separatedanalyte bands may comprise causing the separated analyte bands tomigrate towards an outlet or distal end of the separation channel. Insome instances, the mobilization of a series of one or more separatedanalyte bands may comprise causing the separated analyte bands tomigrate towards an outlet or distal end of the separation channel thatis in fluid communication with a downstream analytical instrument. Insome instances, the outlet or distal end of the separation channel maybe in fluid communication with an electrospray ionization (ESI)interface such that the migrating analyte peaks are injected into a massspectrometer. In some instances, the image data used to detect analytepeak positions and determine analyte pIs may also be used to correlateanalyte separation date with mass spectrometry data. In some instances,the image data used to detect analyte peak positions may be used toyield information on the mobilization reaction and/or to correlate themobilization information with the mass spectrometry data.

In some instances, other characterization techniques may be used tomonitor the one or more separation reactions. In some instances, dynamiclight scattering may be used in at least one of the separation channelswhile performing a separation reaction (e.g., isoelectric focusing). Insome instances, dynamic light scattering may be used to determine a sizedistribution profile, an aggregation state, or a hydrodynamic radius ofone or more analytes. The one or more analytes may be separated using aseparation reaction (e.g., isoelectric focusing).

In preferred aspects, the disclosed methods may be performed in amicrofluidic device format, thereby allowing for processing of extremelysmall sample volumes and integration of two or more sample processingand separation steps. In another preferred aspect, the disclosedmicrofluidic devices comprise an integrated interface for coupling to adownstream analytical instrument, e.g., an ESI interface for performingmass spectrometry on the separated analytes. In some instances, thedisclosed methods may be performed in a more conventional capillaryformat.

Various aspects of the disclosed methods, devices, and systems describedherein may be applied to any of the particular applications set forthbelow. It shall be understood that different aspects of the disclosedmethods, devices, and systems can be appreciated individually,collectively, or in combination with each other.

Definitions: Unless otherwise defined, all of the technical terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art in the field to which this disclosure belongs.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Any reference to “or” herein is intended toencompass “and/or” unless otherwise stated. Similarly, the terms“comprise”, “comprises”, “comprising”, “include”, “includes”, and“including” are not intended to be limiting.

As used herein, the phrases “including, but not limited to . . . ” and“one non-limiting example is . . . ” are meant to be inclusive ofvariations and derivatives of the given example, as commonly understoodby one of ordinary skill in the art in the field to which thisdisclosure belongs.

As used herein, the term ‘about’ a number refers to that number plus orminus 10% of that number. The term ‘about’ when used in the context of arange refers to that range minus 10% of its lowest value and plus 10% ofits greatest value.

As used herein, the terms “characterization” and “analysis” may be usedinterchangeably. To “characterize” or “analyze” may generally mean toassess a sample, for example, to determine one or more properties of thesample or components thereof, or to determine the identity of thesample.

As used herein, the terms “chip” and “device” may be usedinterchangeably herein.

As used herein, the terms “analyte” and “species” may be usedinterchangeably. An analyte generally means a molecule, biomolecule,chemical, macromolecule, etc., that differs from another molecule,biomolecule, chemical, macromolecule, etc. in a measurable property. Forexample, two species may have a slightly different mass, hydrophobicity,charge or net charge, isoelectric point, efficacy, or may differ interms of chemical modifications, protein modifications, etc.

Samples: The disclosed methods, devices, systems, and software may beused for separation and characterization of analytes obtained from anyof a variety of biological or non-biological samples. Examples include,but are not limited to, tissue samples, cell culture samples, wholeblood samples (e.g., venous blood, arterial blood, or capillary bloodsamples), plasma, serum, saliva, interstitial fluid, urine, sweat,tears, protein samples derived from industrial enzyme or biologic drugmanufacturing processes, environmental samples (e.g., air samples, watersamples, soil samples, surface swipe samples), and the like. In someembodiments, the samples may be processed using any of a variety oftechniques known to those of skill in the art prior to analysis usingthe disclosed methods and devices for integrated chemical separation andcharacterization. For example, in some embodiments the samples may beprocessed to extract proteins or nucleic acids. Samples may be collectedfrom any of a variety of sources or subjects, e.g., bacteria, virus,plants, animals, or humans.

Sample volumes: In some instances of the disclosed methods and devices,the use of a microfluidic device format may enable the processing ofvery small sample volumes. In some embodiments, the sample volume loadedinto the device and used for analysis may range from about 0.1 μl toabout 1 ml. In some embodiments, the sample volume loaded into thedevice and used for analysis may be at least 0.1 μl, at least 1 μl, atleast 2.5 μl, at least 5 μl, at least 7.5 μl, at least 10 μl, at least25 μl, at least 50 μl, at least 75 μl, at least 100 μl, at least 250 μl,at least 500 μl, at least 750 μl, or at least 1 ml. In some embodiments,the sample volume loaded into the device and used for analysis may be atmost 1 ml, at most 750 μl, at most 500 μl, at most 250 μl, at most 100μl, at most 75 μl, at most 50 μl, at most 25 μl, at most 10 μl, at most7.5 μl, at most 5 μl, at most 2.5 μl, at most 1 μl, or at most 0.1 μl.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some embodiments the sample volume loaded into the deviceand used for analysis may range from about 5 μl to about 5004 Those ofskill in the art will recognize that sample volume used for analysis mayhave any value within this range, e.g., about 18 μl.

Analytes: In some instances, a sample may comprise a plurality ofanalyte species. In some instances, all or a portion of the analytespecies present in the sample may be enriched prior to or duringanalysis. In some instances, these analytes can be, for example,glycans, carbohydrates, nucleic acid molecules (e.g., DNA, RNA),peptides, polypeptides, recombinant proteins, intact proteins, proteinisoforms, digested proteins, fusion proteins, antibody-drug conjugates,protein-drug conjugates, metabolites or other biologically relevantmolecules. In some instances, these analytes can be small moleculedrugs. In some instances, these analytes can be protein molecules in aprotein mixture, such as a biologic protein pharmaceutical (e.g., enzymepharmaceutical or antibody pharmaceutical) and/or a lysate collectedfrom cells isolated from culture or in vivo.

Microfluidic devices: Disclosed herein are devices designed to perform aplurality of analyte separation reactions in parallel, i.e., within aplurality of separation channels. In some instances, the discloseddevices are microfluidic devices designed to perform a plurality ofanalyte separation reactions in parallel, i.e., within a plurality ofseparation channels within the device. In some instances, themicrofluidic device may be designed to perform one or more differentseparation steps, i.e., a first separation reaction, a second separationreaction, a third separation reaction, and so forth, for a plurality ofanalyte samples in parallel in the same device, i.e., within a pluralityof first separation channels, second separation channels, thirdseparation channels, and so forth, within the device. In a preferredinstance, at least one of the separation steps may comprise isoelectricfocusing, and the device may be designed to perform two or moreisoelectric focusing reactions in parallel, i.e., in two or moreseparation channels within the device. In some instance, the number ofseparation channels, e.g., 4, 6, or 12, may be chosen to coincide withthe layout of microplate wells used as sample and/or reagent sources.

In some instances, the microfluidic devices comprises a substantiallyplanar substrate, wherein the planar substrate comprises a plurality offluid inlets, which fluid inlets or a portion thereof are located at oneor more edges of the planar substrate, and a plurality of separationchannels comprising (i) a first end that is electrically coupled to anelectrode reservoir (e.g., anolyte reservoir) using a first fixture and(ii) a second end that is electrically coupled to another electrodereservoir (e.g., catholyte reservoir) using a second fixture, andwherein one of the first end or the second end of each separationchannel of the plurality of separation channels is in fluidcommunication with a different fluid inlet of the plurality of fluidinlets. In some instances, the first fixture and/or the second fixturecomprises a membrane. In some instances, the first fixture and/or thesecond fixture is a high voltage electrode fixture and optionallyincludes a membrane.

FIG. 1A provides a non-limiting schematic illustration of a microfluidicdevice comprising a four-channel isoelectric focusing design accordingto one aspect of the present disclosure, as will be discussed in moredetail in Example 1 below.

FIG. 1B provides a non-limiting schematic illustration of a fluidchannel network of an exemplary microfluidic device for performing aseparation reaction and comprising an electrospray tip according to asecond aspect of the present disclosure, as will be described in moredetail Example 14 below.

In some instances, the number of separation channels within the devicethat are configured for performing each separation step (e.g., anisoelectric focusing reaction) in parallel may be at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 12, at least 14, at least 16, at least 18, atleast 20, or more than 20. In some instances, the number of separationchannels within the device that are configured for performing eachseparation step in parallel may be at most 20, at most 18, at most 16,at most 14, at most 12, at most 10, at most 9, at most 8, at most 7, atmost 6, at most 5, at most 4, at most 3, or at most 2. Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someinstances the number of separation channels within the device that areconfigured for performing each separation step in parallel may rangefrom about 4 to about 12. Those of skill in the art will recognize thatthe number of separation channels within the device that are configuredfor performing each separation step in parallel may have any valuewithin this range, e.g., about 5.

In some instances, a proximal end of each separation channel of theplurality of separation channels is in fluid communication with adifferent inlet port, so that a different sample or sample aliquot maybe introduced into each separation channel. In some instances, aproximal end of each separation channel in a subset of the plurality ofseparation channels is in fluid communication with the same inlet port,so that the same sample or sample aliquot may be introduced into thesubset of the separation channels. In some instances, a proximal end ofeach separation channel of the plurality of separation channels is influid communication with the distal or outlet end of an upstreamseparation channel.

In some instances, a distal end of each separation channel of theplurality of separation channels is in fluid communication with adifferent outlet port. In some instances, a distal end of eachseparation channel in a subset of the plurality of separation channelsis in fluid communication with the same outlet port. Such an example ofa shared outlet port among the plurality of separation channels may beuseful for removal of contents therein, e.g., to facilitate wastecollection. In some instances, a distal end of each separation channelof the plurality of separation channels is in fluid communication withthe proximal or inlet end of a downstream separation channel.

In some instances, the disclosed microfluidic devices may comprise twoor more integrated electrodes configured to apply a voltage gradientalong a separation channel or interconnecting channel that intersects aseparation channel. In some instances, the device comprises anintegrated pair of electrodes for each separation channel, wherein oneelectrode of each pair is in contact with the proximal end of aseparation channel, and the other electrode is in contact with thedistal end of the separation channel. In some instances, the disclosedmicrofluidic devices may comprise at least two, at least three, at leastfour, at least five, or at least six integrated electrodes perseparation channel. In some instances, the disclosed microfluidicdevices may comprise an integrated pair of electrodes for eachseparation channel at each stage of separation, e.g., first separationchannels, second separation channels, third separation channels, and soforth.

In addition to a plurality of separation channels (e.g., two or morefirst separation channels, two or more second separation channels, twoor more third separation channels, and so forth), the devices ormicrofluidic devices of the present disclosure may comprise a pluralityof inlet ports, outlet ports, sample and/or reagent introductionchannels, interconnecting channels, sample and/or reagent wastechannels, reservoirs (e.g., sample reservoirs, reagent reservoirs, orwaste reservoirs), micropumps, microvalves, vents, traps, filters,membranes, and the like, or any combination thereof.

The disclosed devices and microfluidic devices may be fabricated usingany of a variety of fabrication techniques and materials known to thoseof skill in the art. In some instances, the devices may be fabricated asa series of two or more separate parts, and subsequently eithermechanically clamped or permanently bonded together to form thecompleted device. In some instances, for example, fluid channels (alsosometimes referred to herein as “microchannels”) may be fabricated in afirst layer (e.g., by photolithographic patterning of a glass substrateand wet chemical etching of the channels to the desired depth), and thensealed by bonding a second layer to the first layer, where through holesin the second layer that intersect with the fluid channels provideexternal access to the fluid channels. In some instances, fluid channelsmay be fabricated in a first layer (e.g., by laser cutting of a channelpattern in a suitable polymer film), and then sealed by sandwiching andbonding the first layer between second and third layers, where throughholes in the second layer and/or third layer that intersect with thefluid channels provide external access to the fluid channels. In thelatter example, the thickness of the first layer defines the thickness(or depth) of the fluid channels.

Examples of suitable fabrication techniques include, but are not limitedto, conventional machining, CNC machining, injection molding, 3Dprinting, alignment and lamination of one or more layers of laser- ordie-cut polymer film, or any of a number of microfabrication techniquessuch as photolithography and wet chemical etching, dry etching, deepreactive ion etching, or laser micromachining. In some embodiments, themicrofluidic structures may be 3D printed from an elastomeric material.

The disclosed devices and microfluidic devices may be fabricated usingany of a variety of materials known to those of skill in the art. Ingeneral, the choice of material used will depend on the choice offabrication technique, and vice versa. Examples of suitable materialsinclude, but are not limited to, glass, quartz, fused-silica, silicon,any of a variety of polymers, e.g. polydimethylsiloxane (PDMS;elastomer), polymethylmethacrylate (PMMA), polycarbonate (PC),polystyrene (PS), polypropylene (PP), polyethylene (PE), polyfluorinatedpolyethylene, high density polyethylene (HDPE), polyether ether ketone,polyimide, cyclic olefin polymers (COP), cyclic olefin copolymers (COC),polyethylene terephthalate (PET), polyether ether ketone (PEEK), epoxyresins, a non-stick material such as teflon (polytetrafluoroethylene(PTFE)), a variety of photoresists such as SU8 or any other thick filmphotoresist, or any combination of these materials. In some instances,different layers in a device or microfluidic device comprising multiplelayers may be fabricated from different materials. In some instances, agiven single layer in a device or microfluidic device comprising one ormore layers may be fabricated from two or more different materials.

In some instances, all or a portion of the device or microfluidic devicemay be optically transparent (e.g., transparent to ultraviolet (UV),visible, and/or near-infrared light) to facilitate imaging of theseparation channels and/or other portions of the device. In someinstances, all or a portion of the separation channels are configuredfor imaging, e.g., whole channel imaging. For example, in some instancesthe separation channels may be fabricated in a layer of optically opaquematerial that is sandwiched between two layers of optically transparentmaterial, thereby forming an “optical slit” through which light may betransmitted and/or collected.

In general, the dimensions of fluid channels, sample and/or reagentreservoirs, etc., in the disclosed devices will be optimized to (i)provide fast, accurate, and reproducible separation of samples or samplealiquots comprising analyte mixtures, and (ii) to minimize sample andreagent consumption. In general, the width of fluid channels orreservoirs may be between about 10 μm and about 2 mm. In some instances,the width of fluid channels (or reservoirs) may be at least 10 μm, atleast 25 μm, at least 50 μm at least 100 μm, at least 200 μm, at least300 μm, at least 400 μm, at least 500 μm, at least 750 μm, at least 1mm, at least 1.5 mm, or at least 2 mm. In some instances, the width offluid channels (or reservoirs) may at most 2 mm, at most 1.5 mm, at most1 mm, at most 750 μm, at most 500 μm, at most 400 μm, at most 300 μm, atmost 200 μm, at most 100 μm, at most 50 μm, at most 25 μm, or at most 10μm. Any of the lower and upper values described in this paragraph may becombined to form a range included within the disclosure, for example, insome instances the width of the fluid channels (or reservoirs) may rangefrom about 100 μm to about 1 mm. Those of skill in the art willrecognize that the width of the fluid channels (or reservoirs) may haveany value within this range, for example, about 80 μm.

In general, the depth of the fluid channels (or reservoirs) will bebetween about 1 μm and about 1 mm. In some instances, the depth of thefluid channels (or reservoirs) may be at least 1 μm, at least 5 μm, atleast 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm,at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, atleast 900 μm, or at least 1 mm. In some instances, the depth of thefluid channels (or reservoirs) may be at most 1 mm, at most 900 μm, atmost 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, atmost 40 μm, at most 30 μm, at most 20 μm, at most 10 μm, at most 5 μm,or at most 1 μm. Any of the lower and upper values described in thisparagraph may be combined to form a range included within thedisclosure, for example, in some instances the depth of the fluidchannels (or reservoirs) may range from about 50 μm to about 100 μm.Those of skill in the art will recognize that the depth of the fluidchannels (or reservoirs) may have any value within this range, forexample, about 55 μm.

Cartridges: In some instances, the disclosed devices or systems may beconfigured to be coupled to one another or may be a part of anintegrated unit, such as a cartridge. The cartridge may comprise themicrofluidic device, a substrate comprising a plurality of separationchannels, reservoirs, reagents, membranes, valves, fixtures (e.g., thosedescribed herein, such as membrane-containing high voltage electrodefixtures) securing devices or features (e.g., screws, pins (e.g., pogopins), adhesives, levers, switches, grooves, form-fitting pairs, hooksand loops, latches, threads, clips, clamps, prongs, rings, rubber bands,rivets, grommets, ties, snaps, tapes, vacuum, seals), gaskets, o-rings,electrodes, or a combination thereof. The cartridge may bemonolithically built or may be modular and comprise removable parts. Forinstance, the microfluidic device may be configured to couple removablyto the cartridge. Similarly, the reservoirs, membranes, valves, etc. mayeach be removable from the cartridge. In the case where one or morecomponents may be removable, the cartridge may be configured such thateach of the individual components may be aligned in place withsufficient tolerance by a user. For example, the cartridge may comprisegrooves and pins, such that the microfluidic device may be integrated bysliding the device along the cartridge until the cartridge reaches a pinfor alignment. In some instances, the device may be configured to bepositioned flush with the cartridge or a portion thereof. In someinstances, the device may be positioned into the cartridge such that oneor more inlets, outlets, etc. may be connected (e.g., fluidically and/orelectrically) to a reservoir, electrode, membrane and/or other usefulinterfacing unit. In some instances, the interfacing of the device andthe reservoirs, electrodes, etc. may be performed by a without anyadditional measurement or adjustment from the user. For example, thereservoirs may be configured to receive an electrode which snaps intoplace or is secured via a pogo pin, thereby establishing electricaland/or fluidic communication. It will be appreciated that these exampleconfigurations of the cartridge and device are not meant to be limiting,and that many different configurations of positioning the microfluidicdevice or other component of the cartridge may be achieved. In someinstances, the cartridge may be configured to be a disposable componentof the systems described herein.

In a preferred embodiment, the cartridge may comprise one or morereservoirs that is configured to contain a desired volume of fluid. Insome instances, the reservoir may be capable of containing at leastabout 200 microliters (μL), at least about 300 μL, at least about 400μL, at least about 500 μL, at least about 600 μL, at least about 700 μL,at least about 800 μL, at least about 900 μL, at least about 1milliliter (mL), at least about 1.5 mL, at least about 2 mL, at leastabout 2.5 mL, at least about 3 mL, at least about 3.5 mL, at least about4 mL, at least about 4.5 mL, or at least about 5 mL. In some instances,the reservoir may be capable of containing at most about 5 mL, at mostabout 4.5 mL, at most about 4 mL, at most about 3.5 mL, at most about 3mL, at most about 2.5 mL, at most about 2 mL, at most about 1.5 mL, atmost about 1 mL, at most about 900 μL, at most about 800 μL, at mostabout 700 μL, at most about 600 μL, at most about 500 μL, at most about400 μL, at most about 300 μL, or at most about 200 μL. Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someinstances the reservoir may contain a volume of fluid that may rangefrom about 200 μL to about 2 mL. Those of skill in the art willrecognize that the reservoir fluid volume capacity may have any valuewithin this range, e.g., about 1.8 mL.

In some embodiments, one or more kits may be provided, which maycomprise the cartridge, one or more reagents, and in some instances,instructions for using the kit. The reagents may be stored in thereservoir as a liquid. In some embodiments, the reagents may be dry,e.g., lyophilized, and able to be reconstituted in a solution or buffer.In some cases, the reagents may be separate from the cartridge and maybe provided in the kit.

In some instances, the reservoirs may be controllably coupled (e.g.,electrically, fluidically) to the microfluidic device. For example, thecartridge may comprise one or more valves, which may be used to controlthe flow volumes or rate in the device. In some cases, the cartridge maycomprise a stop-cock valve or a shear valve (e.g., sliding valve orrotating shear valve), which may allow for controlled flow rate duringdelivery of one or more liquid reagents (e.g., mobilization reagents).In some cases, the cartridge may be integrated or interfaced with asyringe pump, which may be used to control the flow rate of liquid intothe device. In some cases, the flow rate may be controlled using apiston, a spring-loaded device, or other mechanical approaches.

In some instances, the cartridge may be configured to accommodatedifferent types or models of devices. For instance, the cartridge may beconfigured to accommodate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, or 100 different types or models of devices.In some cases, the cartridge may comprise ports or connections that caninterface with the channels of the chip (e.g., interface with the inletsand/or outlets of the chip).

Separation and enrichment of analytes: In some instances, the discloseddevices or systems may be configured to perform one or more separationor enrichment steps in which a plurality of analytes in a mixture areseparated and/or concentrated in individual fractions. For example, insome instances the disclosed devices may be configured to perform afirst enrichment step, in which a mixture of analytes in a sample areseparated into and/or enriched as analyte fractions (e.g., analyte peaksor analyte bands) containing a subset of the analyte molecules from theoriginal sample. In some instances, these separated analyte fractionsmay be mobilized and/or eluted, and in some instances, may then besubjected to another downstream separation and/or enrichment step. Insome instances, e.g., following a final separation and/or enrichmentstep, the separated/enriched analyte fractions may be expelled from thedevice for further analysis.

In some instances, the disclosed devices and systems may be configuredto perform one, two, three, four, or five or more separation and/orenrichment steps. In some instances, one or more of the separation orenrichment steps may comprise a solid-phase separation technique, e.g.,reverse-phase HPLC. In some instances, one or more of the separation orenrichment steps may comprise a solution-phase separation and/orenrichment technique, e.g., capillary zone electrophoresis (CZE) orisoelectric focusing (IEF).

The disclosed devices and systems may be configured to perform any of avariety of analyte separation and/or enrichment techniques known tothose of skill in the art, where the separation or enrichment step(s)are performed in at least a first separation channel that is configuredto be imaged in whole or in part so that the separation process may bemonitored as it is performed. For example, in some instances the imagedseparation may be an electrophoretic separation comprising, e.g.,isoelectric focusing, capillary gel electrophoresis, capillary zoneelectrophoresis, isotachophoresis, capillary electrokineticchromatography, micellar electrokinetic chromatography, flowcounterbalanced capillary electrophoresis, electric field gradientfocusing, dynamic field gradient focusing, and the like, that producesone or more separated analyte fractions from an analyte mixture. In someinstances, a separation and mobilization step may be performed in atleast a first separation channel that is configured to be imaged inwhole or in part so that the separation and mobilization processes maybe monitored as they are performed. In any of these instances, theimaging of the separation channel in whole or in part may be performedcontinuously or intermittently and may be performed prior to, during, orfollowing the separation and/or enrichment process.

In some instances, the use of a microfluidic device format may providefor fast separation times and accurate, reproducible separation data.For example, in instances where the microfluidic device is configured toperform electrophoretic separations and/or isoelectric focusingreactions, the high surface area-to-volume ratios of microfluidicchannels may allow one to use high electric field strengths withoutincurring significant Joule heating, thereby enabling very fastseparation reactions without substantial dispersion and loss ofseparation resolution. In some instances, the precise control of fluidchannel geometries provides for accurate and reproducible control ofsample injection volumes, electric field strengths, etc., therebyenabling very accurate determinations of one or more parameters of theassay, e.g., separation resolution and/or pI determinations.

The one or more parameters of the assay may comprise a characteristic ofthe separation. For example, the one or more parameters may be selectedfrom the group consisting of separation resolution, peak width, peakcapacity, linearity of the pH gradient, and minimum resolvable pIdifference.

In general, the separation time required to achieve complete separationwill vary depending on the specific separation technique and operationalparameters utilized (e.g., separation channel length, microfluidicdevice design, buffer compositions, applied voltages, etc.). In someinstances, the separation times achieved using the disclosed devices andsystems may range from about 0.1 minutes to about 30 minutes. In someinstances, the separation time may be at least 0.1 minutes, at least 0.5minutes, at least 1 minute, at least 5 minutes, at least 10 minutes, atleast 15 minutes, at least 20 minutes, at least 25 minutes, or at least30 minutes. In some instances, the separation time may be at most 30minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, atmost 10 minutes, at most 5 minutes, at most 1 minute, at most 0.5minutes, or at most 0.1 minutes. Any of the lower and upper valuesdescribed in this paragraph may be combined to form a range includedwithin the present disclosure, for example, in some instances theseparation time may range from about 1 minute to about 20 minutes. Thoseof skill in the art will recognize that the separation time may have anyvalue within this range, e.g., about 11.2 minutes.

Similarly, the separation efficiency and resolution achieved using thedisclosed devices and systems may vary depending on the specificseparation technique and operational parameters utilized (e.g.,separation channel length, microfluidic device design, buffercompositions, applied voltages, etc.), as well as whether one or twodimensions of separation are utilized. In some instances, for examplewhen performing isoelectric focusing, the use of switchable electrodesto trigger electrophoretic introduction of a mobilization electrolyteinto the separation channel may result in improved separationresolution. For example, in some instances, the separation resolution ofIEF performed using the disclosed methods and devices may provide for aresolution of analyte bands differing in pI ranging from about 0.1 toabout 0.0001 pH units. In some instances, the IEF separation resolutionmay allow for resolution of analyte bands differing in pI by less than0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001,less than 0.0005, or less than 0.0001 pH units.

Accordingly, in some instances, e.g., when using imaging of all or aportion of a separation channel to identify the positions of pI markersin an isoelectric focusing reaction and determine a pI value forseparated analytes, the accuracy with which the pI value may bedetermined may be less than ±0.1 pH unit, less than ±0.05 pH units, lessthan ±0.01 pH units, less than ±0.005 pH units, less than ±0.001 pHunits, less than ±0.0005 pH units, or less than ±0.0001 pH units.

In some instances, the peak capacity achieved using the discloseddevices may range from about 100 to about 20,000. In some instances, thepeak capacity may be at least 100, at least 200, at least 300, at least400, at least 500, at least 600, at least 700, at least 800, at least900, at least 1,000, at least 2,000, at least 3,000, at least 4,000, atleast 5,000, at least 10,000, at least 15,000, or at least 20,000. Insome instances, the peak capacity may be at most 20,000, at most 15,000,at most 10,000, a most 5,000, at most 4,000, at most 3,000, at most2,000, at most 1,000, at most 900, at most 800, at most 700, at most600, at most 500, at most 400, at most 300, at most 200, or at most 100.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the peak capacity may range from about 400 toabout 2,000. Those of skill in the art will recognize that the peakcapacity may have any value within this range, e.g., about 285.

Capillary isoelectric focusing (CIEF): In some embodiments, theseparation technique may comprise isoelectric focusing (IEF), e.g.,capillary isoelectric focusing (CIEF). Isoelectric focusing (or“electrofocusing”) is a technique for separating molecules bydifferences in their isoelectric point (pI), i.e., the pH at which themolecules have a net zero charge. CIEF involves adding ampholyte(amphoteric electrolyte) solutions to a sample channel between reagentreservoirs containing an anode or a cathode to generate a pH gradientwithin a separation channel (i.e., the fluid channel connecting theelectrode-containing wells, e.g., the lumen of a capillary or a channelin a microfluidic device) across which a separation voltage is applied.The ampholytes can be solution phase or immobilized on the surface ofthe channel wall. Negatively charged molecules migrate through the pHgradient in the medium toward the positive electrode while positivelycharged molecules move toward the negative electrode. A protein (orother molecule) that is in a pH region below its isoelectric point (pI)will be positively charged and so will migrate towards the cathode(i.e., the negatively charged electrode). The protein's overall netcharge will decrease as it migrates through a gradient of increasing pH(due, for example, to protonation of carboxyl groups or other negativelycharged functional groups) until it reaches the pH region thatcorresponds to its pI, at which point it has no net charge and somigration ceases. As a result, a mixture of proteins separates based ontheir relative content of acidic and basic residues and becomes focusedinto sharp stationary bands with each protein positioned at a point inthe pH gradient corresponding to its pI. The technique is capable ofextremely high resolution, with proteins differing by a single chargebeing fractionated into separate bands. In some embodiments, isoelectricfocusing may be performed in a separation channel that has beenpermanently or dynamically coated, e.g., with a neutral and hydrophilicpolymer coating, to eliminate electroosmotic flow (EOF). Examples ofsuitable coatings include, but are not limited to, amino modifiers,hydroxypropylcellulose (HPC) and polyvinylalcohol (PVA), Guarant® (AlcorBioseparations), linear polyacrylamide, polyacrylamide, dimethylacrylamide, polyvinylpyrrolidine (PVP), methylcellulose,hydroxyethylcellulose (HEC), hydroxyprpylmethylcellulose (HPMC),triethylamine, proylamine, morpholine, diethanolamine, triethanolamine,diaminopropane, ethylenediamine, chitosan, polyethyleneimine,cadaverine, putrescine, spermidine, diethylenetriamine,tetraethylenepentamine, cellulose, dextran, polyethylene oxide (PEO),cellulose acetate, amylopectin, ethylpyrrolidine methacrylate, dimethylmethacrylate, didodecyldimethylammonium bromide, Brij 35, sulfobetains,1,2-dilauryloylsn-phosphatidylcholine,1,4-didecyl-1,4-diazoniabicyclo[2,2,2]octane dibromide, agarose,poly(Nhydroxyethylacrylamide), pole-323, hyperbranched polyamino esters,pullalan, glycerol, adsorbed coatings, covalent coatings, dynamiccoatings, etc. In some embodiments, isoelectric focusing may beperformed (e.g., in uncoated separation channel) using additives such asmethylcellulose, glycerol, urea, formamide, surfactants (e.g., Triton-X100, CHAPS, digitonin) in the separation medium to significantlydecrease the electroosmotic flow, allow better protein solubilization,and limit diffusion inside the capillary (e.g., in the lumen of thecapillary) or fluid channel by increasing the viscosity of theelectrolyte.

As noted above, the pH gradient used for capillary isoelectric focusingtechniques is generated through the use of ampholytes, i.e., amphotericmolecules that contain both acidic and basic groups and that existmostly as zwitterions within a certain range of pH. The portion of theelectrolyte solution on the anode side of the separation channel isknown as an “anolyte”. That portion of the electrolyte solution on thecathode side of the separation channel is known as a “catholyte”. Avariety of electrolytes may be used in the disclosed methods and devicesincluding, but not limited to, phosphoric acid, sodium hydroxide,ammonium hydroxide, glutamic acid, lysine, formic acid, dimethylamine,triethylamine, acetic acid, piperidine, diethylamine, and/or anycombination thereof. The electrolytes may be used at any suitableconcentration, such as 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, etc. The concentration of the electrolytesmay be at least 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%. The concentration of the electrolytes may be atmost 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, 0.01%,0.001%, 0.0001%. A range of concentrations of the electrolytes may beused, e.g., 0.1%-2%. Ampholytes can be selected from any commercial ornon-commercial carrier ampholytes mixtures (e.g., Servalyt pH 4-9(Serva, Heildelberg, Germany), Beckman pH 3-10 (Beckman Instruments,Fullerton, Calif., USA), Ampholine 3.5-9.5 and Pharmalyte 3-10 (bothfrom General Electric Healthcare, Orsay, France), AESlytes (AES), FLUKAampholyte (Thomas Scientific, Swedesboro, N.J.), Biolyte (Bio-Rad,Hercules, Calif.)), and the like. Carrier ampholyte mixtures maycomprise mixtures of small molecules (about 300-1,000 Da) containingmultiple aliphatic amino and carboxylate groups that have closely spacedpI values and good buffering capacity. In the presence of an appliedelectric field, carrier ampholytes partition into smooth linear ornon-linear pH gradients that increase progressively from the anode tothe cathode.

Any of a variety of pI standards may be used in the disclosed methodsand devices for calculating the isoelectric point for separated analytepeaks. For example, pI markers generally used in CLEF applications,e.g., protein pI markers and synthetic small molecule pI markers, may beused. In some instances, protein pI markers may be specific proteinswith commonly accepted pI values. In some instances, the pI markers maybe detectable, e.g., via imaging. A variety or combination of protein pImarkers or synthetic small molecule pI markers that are commerciallyavailable, e.g., the small molecule pI markers available from AdvancedElectrophoresis Solutions, Ltd. (Cambridge, Ontario, Canada),ProteinSimple, the peptide library designed by Shimura, and Slais dyes(Alcor Biosepartions), may be used.

Capillary zone electrophoresis (CZE): In some instances, the separationor enrichment technique may comprise capillary zone electrophoresis, amethod for separation of charged analytes in solution in an appliedelectric field. The net velocity of charged analyte molecules isinfluenced both by the electroosmotic flow (EOF), μEOF, exhibited by theseparation system and the electrophoretic mobility, μEP, for theindividual analyte (dependent on the molecule's size, shape, andcharge), such that analyte molecules exhibiting different size, shape,or charge exhibit differential migration velocities and separate intobands. In contrast to other capillary electrophoresis methods, CZE uses“simple” buffer, or background electrolyte, solutions for separation.

Capillary gel electrophoresis (CGE): In some instances, the separationor enrichment technique may comprise capillary gel electrophoresis, amethod for separation and analysis of macromolecules (e.g., DNA, RNA andproteins) and their fragments based on their size and charge. The methodcomprises use of a gel-filled separation channel, where the gel acts asan anti-convective and/or sieving medium during electrophoretic movementof charged analyte molecules in an applied electric field. The gelfunctions to suppress thermal convection caused by application of theelectric field, and also acts as a sieving medium that retards thepassage of molecules, thereby resulting in a differential migrationvelocity for molecules of different size or charge.

Capillary isotachophoresis (CITP): In some instances, the separationtechnique may comprise capillary isotachophoresis, a method forseparation of charged analytes that uses a discontinuous system of twoelectrolytes (known as the leading electrolyte and the terminatingelectrolyte) within a capillary or fluid channel of suitable dimensions.The leading electrolyte contains ions with the highest electrophoreticmobility, while the terminating electrolyte contains ion with the lowestelectrophoretic mobility. The analyte mixture (i.e., the sample) to beseparated is sandwiched between these two electrolytes, and applicationof an electric field results in partitioning of the charged analytemolecules within the capillary or fluid channel into closely contiguouszones in order of decreasing electrophoretic mobility. The zones movewith constant velocity in the applied electric field such that adetector, e.g., a conductivity detector, photodetector, or imagingdevice, may be utilized record their passage along the separationchannel. Unlike capillary zone electrophoresis, simultaneousdetermination or detection of anionic and cationic analytes is notfeasible in a single analysis performed using capillaryisotachophoresis.

Capillary electrokinetic chromatography (CEC): In some instances, theseparation technique may comprise capillary electrokineticchromatography, a method for separation of analyte mixtures based on acombination of liquid chromatographic and electrophoretic separationmethods. CEC offers both the efficiency of capillary electrophoresis(CE) and the selectivity and sample capacity of packed capillaryhigh-performance liquid chromatography (HPLC). Because the capillariesused in CEC are packed with HPLC packing materials, the wide variety ofanalyte selectivity's available in HPLC are also available in CEC. Thehigh surface area of these packing materials enables CEC capillaries toaccommodate relatively large amounts of sample, making detection of thesubsequently eluted analytes a somewhat simpler task than it is incapillary zone electrophoresis (CZE).

Micellar electrokinetic chromatography (MEKC): In some instances, theseparation technique may comprise capillary electrokineticchromatography, a method for separation of analyte mixtures based ondifferential partitioning between surfactant micelles (apseudo-stationary phase) and a surrounding aqueous buffer solution (amobile phase). The basic set-up and detection methods used for MEKC arethe same as those used in CZE. The difference is that the buffersolution contains a surfactant at a concentration that is greater thanthe critical micelle concentration (CMC), such that surfactant monomersare in equilibrium with micelles. MEKC is typically performed in opencapillaries or fluid channels using alkaline conditions to generate astrong electroosmotic flow. Sodium dodecyl sulfate (SDS) is one exampleof a commonly used surfactant in MEKC applications. The anioniccharacter of the sulfate groups of SDS cause the surfactant and micellesto have electrophoretic mobility that is counter to the direction of thestrong electroosmotic flow. As a result, the surfactant monomers andmicelles migrate quite slowly, though their net movement is still in thedirection of the electroosmotic flow, i.e., toward the cathode. DuringMEKC separations, analytes distribute themselves between the hydrophobicinterior of the micelle and hydrophilic buffer solution. Hydrophilicanalytes that are insoluble in the micelle interior migrate at theelectroosmotic flow velocity, u_(o), and will be detected at theretention time of the buffer, t_(M). Hydrophobic analytes thatsolubilize completely within the micelles migrate at the micellevelocity, u_(c), and elute at the final elution time, t_(c).

Flow counterbalanced capillary electrophoresis (FCCE): In someinstances, the separation technique may comprise flow counterbalancedcapillary electrophoresis, a method for increasing the efficiency andresolving power of capillary electrophoresis that utilizes apressure-induced counter-flow to actively retard, halt, or reverse theelectrokinetic migration of an analyte through a capillary. Byretarding, halting, or moving the analytes back and forth across adetection window, the analytes of interest are effectively confined tothe separation channel for much longer periods of time than under normalseparation conditions, thereby increasing both the efficiency and theresolving power of the separation.

Chromatography: In some instances, the separation technique may comprisea chromatographic technique in which the analyte mixture in the samplefluid (the mobile phase) is passed through a column or channel-packingmaterial (the stationary phase) which differentially retains the variousconstituents of the mixture, thereby causing them to travel at differentspeeds and separate. In some instances, a subsequent step of elution ormobilization may be required to displace analytes that have a highbinding affinity for the stationary phase. Examples of chromatographictechniques the may be incorporated into the disclosed methods include,but are not limited to, ion exchange chromatography, size-exclusionchromatography, and reverse-phase chromatography.

Mobilization of separated analyte species: In some instances, providedherein are devices and systems configured to perform, e.g., achromatographic separation technique such as reverse-phasechromatography. The method implemented by the device or system mayfurther comprise elution of the analyte species retained on thestationary phase in each of a plurality of separation channels (e.g., bysimultaneously or independently changing a buffer that flows througheach a plurality of separation channels), which may be referred to as a“mobilization” step or reaction. In some instances, the methodimplemented by the device or system may further comprise simultaneouslyor independently applying pressure to each of a plurality of separationchannels, or simultaneously or independently introducing an electrolyteinto each of a plurality of separation channels to disrupt the pHgradient used for isoelectric focusing, and thus trigger migration ofthe separated analyte peaks out of the separation channels, which mayalso be referred to as a “mobilization” step. In some instances, theforce used to drive the separation reactions (e.g., pressure forreverse-phase chromatography, or an electric field for electrokineticseparation or isoelectric focusing reactions) may be turned off duringthe mobilization step. In some instances, the force used to drive theseparation reactions may be left on during the mobilization step. Insome instances of the disclosed methods, e.g., those comprising anisoelectric focusing step, the separated analyte bands may be mobilized(e.g., using hydrodynamic pressure and/or a chemical mobilizationtechnique) such that the separated analyte bands migrate towards an endof each of a plurality of separation channels that is connected toanother fluid channel (which may be, e.g., an outlet, a waste reservoir,or a second separation channel). In some instances, e.g., in thoseinstances where capillary gel electrophoresis, capillary zoneelectrophoresis, isotachophoresis, capillary electrokineticchromatography, micellar electrokinetic chromatography, flowcounterbalanced capillary electrophoresis, or any other separationtechnique that separates components of an analyte mixture bydifferential velocity is employed, the separation step itself may beviewed as a mobilization step.

In some instances, mobilization of the analyte bands may be implementedby simultaneously or independently applying hydrodynamic pressure to oneend of each the plurality of separation channels. In some instances,mobilization of the analyte bands may be implemented by orienting thedevice such that the plurality of separation channels is in a verticalposition so that gravity may be employed. In some instances,mobilization of the analyte bands may be implemented using EOF-assistedmobilization. In some instances, mobilization of the analyte bands maybe implemented using chemical mobilization, e.g., by simultaneously orindependently introducing a mobilization electrolyte into each of theplurality of separation channels that shifts the local pH in a pHgradient used for isoelectric focusing. In some instances, anycombination of these mobilization techniques may be employed.

In one preferred instance, the mobilization step forisoelectrically-focused analyte bands comprises chemical mobilization.Compared with pressure-based mobilization, chemical mobilization has theadvantage of exhibiting minimal band broadening by overcoming thehydrodynamic parabolic flow profile induced through the use of pressure.Chemical mobilization may be implemented by introducing an electrolyte(i.e., a “mobilization electrolyte”) into the separation channel toalter the local pH and/or net charge on separated analyte bands (orzwitterionic buffer components) such that they (or the zwitterionicbuffer components and associated hydration shells) migrate in an appliedelectric field. In some instances, the polarity of the applied electricfield used to mobilize separated analyte bands may be such that analytesmigrate towards an anode that is in electrical communication with theoutlet or distal end of the separation channel (anodic mobilization). Insome instances, the polarity of the applied electric field used tomobilize separated analyte bands may be such that analytes migratetowards a cathode that is in electrical communication with the outlet ordistal end of the separation channel (cathodic mobilization).Mobilization electrolytes comprise either anions or cations that competewith hydroxyls (cathodic mobilization) or hydronium ions (anodicmobilization) for introduction into the separation channel or capillary.Examples of bases that may be used as catholytes for anodic mobilizationinclude, but are not limited to, sodium hydroxide, ammonium hydroxide(“ammonia”), diethylamine, dimethyl amine, piperidine, etc. Examples ofacids that may be used as anolytes in cathodic mobilization include, butare not limited to, phosphoric acid, acetic acid, formic acid, andcarbonic acid, etc. In some instances, mobilization may be initiated bythe addition of salts (e.g., sodium chloride) to the anolyte orcatholyte. In some instances, an anode may be held at ground, and anegative voltage is applied to the cathode. In some instances, a cathodemay be held at ground, and a positive voltage is applied to the anode.In some instances, a non-zero negative voltage may be applied to thecathode, and a non-zero positive voltage may be applied to the anode. Insome instances, a non-zero positive voltage may be applied to both theanode and the cathode. In some instances, a non-zero negative voltagemay be applied to both the anode and the cathode.

In some instances, mobilization of separated analyte bands may beinitiated at a user-specified time point that triggers switchableelectrodes (e.g., a cathode in electrical communication with the distalend of each of the plurality of separation channels, and a cathode inelectrical communication with a proximal end of each of a plurality ofmobilization channels (e.g., fluid channels that intersects theseparation channels near the outlet or distal end of each separationchannel)) between on and off states to control the electrophoreticintroduction of a mobilization buffer or electrolyte into a separationchannel.

In some instances, a user-specified time for independently triggering atransition of one, two, or three or more switchable electrodes betweenon and off states for each of the plurality of separation channels mayrange from about 30 seconds, to about 30 minutes for any of themobilization schemes. In some instances, the user-specified time may beat least 30 second, at least 1 minute, at least 2 minutes, at least 3minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, atleast 15 minutes, at least 20 minutes, at least 25 minutes, or at least30 minutes. In some instances, the user-specified time may be at most 30minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, atmost 10 minutes, at most 5 minutes, at most 4 minutes, at most 3minutes, at most 2 minutes, at most 1 minute, or at most 30 seconds. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the user-specified time may range from about2 minutes to about 25 minutes. Those of skill in the art will recognizethat the user-specified time may have any value within this range, e.g.,about 8.5 minutes.

In some instances, the electric field used to effect mobilization in anyof the mobilization scenarios disclosed herein (or to performelectrokinetic separation or isoelectric focusing reactions in thoseinstances where such separation techniques are performed) may range fromabout 0 V/cm to about 1,000 V/cm. In some instances, the electric fieldstrength may be at least 0 V/cm, at least 20 V/cm, at least 40 V/cm, atleast 60 V/cm, at least 80 V/cm, at least 100 V/cm, at least 150 V/cm,at least 200 V/cm, at least 250 V/cm, at least 300 V/cm, at least 350V/cm, at least 400 V/cm, at least 450 V/cm, at least 500 V/cm, at least600 V/cm, at least 700 V/cm, at least 800 V/cm, at least 900 V/cm, or atleast 1,000 V/cm. In some instances, the electric field strength may beat most 1,000 V/cm, at most 900 V/cm, at most 800 V/cm, at most 700V/cm, at most 600 V/cm, at most 500 V/cm, at most 450 V/cm, at most 400V/cm, at most 350 V/cm, at most 300 V/cm, at most 250 V/cm, at most 200V/cm, at most 150 V/cm, at most 100 V/cm, at most 80 V/cm, at most 60V/cm, at most 40 V/cm, at most 20 V/cm, or at most 0 V/cm. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the electric field strength time may range from about 40V/cm to about 650 V/cm. Those of skill in the art will recognize thatthe electric field strength may have any value within this range, e.g.,about 575 V/cm.

In some instances, mobilization of separated analyte bands may beinitiated based on data derived from independently monitoring thecurrent (or conductivity) for each of the plurality of separationchannels where, for example, in the case of isoelectric focusing thecurrent passing through a separation channel may reach a minimum value.In some instances, the detection of a minimum current value, or acurrent value that remains constant or below a specified threshold for aspecified period of time, may be used to determine if an isoelectricfocusing reaction has reached completion and may thus be used to triggerthe initiation of a chemical mobilization step.

In some instances, the minimum current value or threshold current valuemay range from about 0 μA to about 100 μA. In some instances, theminimum current value or threshold current value may be at least 0 μA,at least 1 μA, at least 2 μA, at least 3 μA, at least 4 μA, at least 5μA, at least 10 μA, at least 20 μA, at least 30 μA, at least 40 μA, atleast 50 μA, at least 60 μA, at least 70 μA, at least 80 μA, at least 90μA, or at least 100 μA. In some instances, the minimum current value orthreshold current value may be at most 100 μA, at most 90 μA, at most 80μA, at most 70 μA, at most 60 μA, at most 50 μA, at most 40 μA, at most30 μA, at most 20 μA, at most 10 μA, at most 5 μA, at most 4 μA, at most3 μA, at most 2 μA, at most 1 μA, or at most 0 μA. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe minimum current value or threshold current value may range fromabout 10 μA to about 90 μA. Those of skill in the art will recognizethat the minimum current value or threshold current value may have anyvalue within this range, e.g., about 16 μA.

In some instances, the specified period of time may be at least 5seconds, at least 10 seconds, at least 15 seconds, at least 20 seconds,at least 25 seconds, at least 30 seconds, at least 35 seconds, at least40 seconds, at least 45 seconds, at least 50 seconds, at least 55seconds, or at least 60 seconds. In some instances, the specified periodof time may be at most about 60 seconds, at most about 55 seconds, atmost about 50 seconds, at most about 45 seconds, at most about 40seconds, at most about 35 seconds, at most about 30 seconds, at mostabout 25 seconds, at most about 20 seconds, at most about 15 seconds, atmost about 10 seconds, or at most about 5 seconds. Amy of the lower andupper values described herein may be combined to form a range includedwithin the present disclosure, in some instances the specified period oftime may range from about 5 seconds to about 30 seconds. Those of skillin the art will recognize that the specified period of time may have anyvalue within this range, e.g., about 32 seconds.

In some instances, mobilization of separated analyte bands may beinitiated based on data derived from images (e.g., by performingautomated image processing) of the plurality of separation channels asseparation reactions are performed. The image-derived data may be usedto monitor the presence or absence of one or more analyte peaks, thepositions of one or more analyte peaks, the widths of one or moreanalyte peaks, the velocities of one or more analyte peaks, separationresolution, a rate of change or lack thereof in the presence, position,width, or velocity of one or more analyte peaks, or any combinationthereof, and may be used to determine whether a separation reaction iscomplete and/or to trigger the initiation of a mobilization step in agiven separation channel. In some cases, completion of a separation stepmay be determined by monitoring the rate of change of a separationperformance parameter (e.g., peak position or peak width) over a periodof time (e.g., over a period of 10 to 60 seconds).

In one preferred aspect of the disclosed methods, a chemicalmobilization step may be initiated within a microfluidic device designedto integrate CIEF with ESI-MS by changing an electric field within thedevice to electrophorese a mobilization electrolyte into the separationchannel. In some instances, the initiation of the mobilization step maybe triggered based on data derived from images of all or a portion ofthe separation channel. In some instances, the change in electric fieldmay be implemented by connecting or disconnecting one or more electrodesattached to one or more power supplies, wherein the one or moreelectrodes are positioned in reagent wells on the device or integratedwith fluid channels of the device. In some instances, the connecting ordisconnecting of one or more electrodes may be controlled using acomputer-implemented method and programmable switches, such that thetiming and duration of the mobilization step may be coordinated with theseparation step. In some instances, changing an electric field withinthe device may be used to electrophoretically or electro-osmoticallyflow a mobilization buffer into a separation channel comprising astationary phase such that retained analytes are released from thestationary phase.

In some instances, three or more electrodes for each separation channelmay be connected to or integrated into the device. For example, a firstelectrode may be coupled electrically to a proximal end of theseparation channel. Similarly, a second electrode may then be coupled tothe distal end of the separation channel, and a third electrode may becoupled with a mobilization channel that intersects with the separationchannel, e.g., at a distal end of the separation channel, and thatconnects to or comprises a reservoir containing mobilization buffers.Upon completion of the separation step, as determined by image-basedmethods, the electric coupling of the second or third electrodes withtheir respective channels may be switchable between “on” and “off”states. In one such an example, the second electrode that forms theanode or cathode of the separation circuit may switch to an “off” mode,and the third electrode, which may be off during the separation, mayswitch to an “on” mode, to initiate introduction of mobilization bufferinto the channel (e.g., via electrophoresis). In some instances, “on”and “off” states may comprise complete connection or disconnection ofthe electrical coupling between an electrode and a fluid channelrespectively. In some instances, “on” and “off” states may compriseclamping the current passing through a specified electrode to non-zeroor zero microamperes respectively.

In some instances, triggering or initiation of a mobilization step maycomprise detecting no change or a change of less than a specifiedthreshold for one or more image-derived separation parameters asdescribed above. For example, in some instances a change of less than20%, 15%, 10%, or 5% in one or more image-derived parameters (e.g., peakposition, peak width, peak velocity, etc.) may be used to trigger themobilization step.

In some instances, triggering or initiation of a mobilization step maycomprise detecting no change or a rate of change of less than aspecified threshold for one or more image-derived separation parametersas described above. For example, in some instances a change of less than20%, 15%, 10%, or 5% in one or more image-derived parameters (e.g., peakposition, peak width, peak velocity, etc.) over a time period of atleast 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, or 60 seconds(or any combination of these percentage changes and time periods) may beused to trigger the mobilization step.

In some instances, a calibrant may be used during the mobilization stepto correlate and/or calibrate information from the mass spectrometer. Insome instances, the calibrant may comprise a peptide, a polypeptide, aprotein, or other molecule (either natural or synthetic) with a knownmass. In some instances, the calibrant will be mixed with the mobilizersolution. The calibrant may be used to calibrate the mass spectrometer.In some instances, the calibrant may be used to correlate informationfrom the mass spectrometer to the mobilization process or the separationprocess. For example, the calibrant may be monitored during theseparation (e.g., isoelectric focusing) or mobilization.

Altering high and low separation/mobilization voltage to keep ESI tipvoltage constant: In some embodiments, the ESI ion source on the massspectrometer will have an adjustable power supply capable of setting anegative voltage on the mass spectrometer. In some embodiments, the ESIion source on the mass spectrometer will have an adjustable power supplycapable of setting a positive voltage on the mass spectrometer. In someembodiments, the ESI ion source on the mass spectrometer will be held atground. In some embodiments, the ESI tip on the capillary ormicrofluidic device will be held at or close to ground to generate anelectric field between the ESI tip and the charged ESI ion source on themass spectrometer. In some embodiments, the ESI tip on the capillary ormicrofluidic device will be held at a positive or negative voltage togenerate an electric field between the ESI tip and the grounded ESI ionsource on the mass spectrometer.

FIG. 36 provides an exemplary flowchart of a computer-controlledfeedback loop to maintain a constant voltage drop of 3000V between theanode and cathode while keeping the ESI tip voltage at 0V duringmobilization. In some embodiments, this feedback loop may be implementedwhen the mass spectrometer ESI ion source is set at a positive ornegative voltage relative to ground (for example, −3500V). In thisexample, ΔV between anolyte port 108 and mobilizer port 104 is kept at3000V by initially setting anolyte port 108 at +3000V and mobilizer port104 at 0V in FIG. 1B. In some embodiments, a different ΔV may be set bysetting anolyte port 108 to a different value. In some embodiments,anodic mobilization may be used, and port 108 would be a catholyte port,set to, for example, −3000V. In the example outlined in FIG. 36, duringmobilization, the resistance in separation channel 112 is dropping dueto analyte and ampholytes in the separation regaining charge. Thiscauses the voltage drop across channel 112 to drop, leading to anincrease in voltage at ESI tip 116, according to equation 1:V ₁₁₆=(ΔV ¹⁰⁸⁻¹⁰⁴)*(R ₁₀₅)/(R ₁₀₉ +R ₁₁₂ +R ₁₀₅)However, by measuring or calculating ESI tip voltage 116, the voltagesettings at anolyte port 108 and mobilizer port 104 can be adjusted. Bysubtracting ESI tip voltage 116 from both anolyte port 108 and mobilizerport 104 settings, ΔV¹¹⁰⁻¹⁰⁴ remains 3000V so the mobilization isunaffected, but ESI tip 116 voltage is set to 0 according to equation 2:V ₁₁₆=(ΔV ¹⁰⁸⁻¹⁰⁴)*(R ₁₀₅)/(R ₁₀₉ +R ₁₁₂ +R ₁₀₅)+V ₁₀₄This feedback loop continues to operate until the mobilization iscomplete, adjusting ESI tip 116 voltage to 0 at a regular frequency,e.g., the Nyquist rate, or about 0.2 Hz. In some instances, the voltageat ESI tip 116 may be adjusted to 0 at a rate of at least 0.01 Hz, 0.1Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1Hz, 10 Hz, 100 Hz, or 1,000 Hz. Maintaining a constant stable voltage atESI tip 116 can be critical to maintaining stable electrospray duringthe mobilization process.

In some instances, the feedback loop operates to maintain the voltage atthe ESI tip to within a specified percentage of a pre-set value. Forexample, in some instances, the feedback loop operates to maintain thevoltage at the ESI tip to within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, or 0.1% of a pre-set value.

In some embodiments, the mass spectrometer ESI ion source is held atground, and ESI tip 116 will need to be kept at a constant positive ornegative voltage in order to create an electric field between ESI tip116 and the mass spectrometer. In some embodiments, ESI tip voltage(e.g., the pre-set value) may be around +5000V, around +4000V, around+3500V, around +3000V, around +2500V, around +2000V, around +1500Varound +1000V, around +500V, or around −5000V, around −4000V, around−3500V, around −3000V, around −2500V, around −2000V, around −1500V,around −1000V, or around −500V. FIG. 37 provides an example flowchart ofa computer-controlled feedback loop to maintain a constant voltage dropof 3000V between the anode and cathode while keeping the ESI tip voltagepotential at 3000V during mobilization. Operation of thecomputer-controlled feedback loop is the same as in FIG. 36, exceptvoltages at anolyte port 108 and mobilizer port 104 are offset by+3000V, which offsets the voltage at ESI tip 116 to +3000V, stillobeying equation 2. In some embodiments control of the electric fieldstrength can be accomplished using analog circuitry. In someembodiments, the control of voltages at one or more electrodes incontact with the capillary-based or microfluidic device-based separationsystem may be provided by using one, two, three, or four or moreindependent high-voltage power supplies. In some instance, the controlof voltages at one or more electrodes in contact with thecapillary-based or microfluidic device-based separation system may beprovided, e.g., by using a single, multiplexed high-voltage powersupply.

In some instances, the feedback loop operates to maintain the electricfield strength within the separation channel, or the voltage dropbetween the anode and cathode, to within a specified percentage of apre-set value. For example, in some instances, the feedback loopoperates to maintain the electric field strength within the separationchannel, or the voltage drop between the anode and cathode, to within10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01% of apre-set value. In some instances, the feedback loop operates to maintainthe ESI tip voltage to within 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V,or 1V of a pre-set value.

In some embodiments, an alternating current (AC) signal generator suchas a lock-in amplifier, function generator, oscillator, or other ACsignal generator may be electrically coupled to a pair of electrodes. Insome embodiments, the AC signal generator may be a SR8-30 (StanfordResearch Systems) lock-in amplifier. In some embodiments, a plurality ofAC signal generators may be electrically coupled to multiple pairs ofelectrodes. In some embodiments, the AC signal generator may beconfigured to superimpose an AC voltage on a DC voltage set between twoelectrodes. In some embodiments, the AC current created by the AC signalgenerator may be measured. In some embodiments, the AC current createdby the AC signal generator may be used to calculate the resistance in amicrofluidic channel. In some embodiments, the resistance of themicrofluidic channel may be changing over time. In some embodiments, theresistance of the microfluidic channel may be changing over time due toisoelectric focusing. In some embodiments, the resistance of themicrofluidic channel may be changing over time due to chemicalmobilization. In some embodiments, the resistance of the microfluidicchannel may be changing over time due to introduction of new reagentinto a channel network between a pair of electrodes. In someembodiments, the AC signal generator may be connected to an electrode inelectrical communication with a distal end of a separation channel andan electrode in electrical communication with a proximal end of the sameseparation channel. In some embodiments, a change in resistance in amicrofluidic channel may be measured. In some embodiments, a measuredchange in resistance in a microfluidic channel may be used to maintain aconstant voltage potential within the fluidic network. In someembodiments, the frequency of the AC signal generated may be at least0.05 Hz, at least 0.1 Hz, at least 0.5 Hz, at least 1 Hz, at least 5 Hz,at least 10 Hz, at least 50 Hz, at least 100 Hz, at least 500 Hz, atleast 1000 Hz, at least 5 kHz, at least 10 kHz, at least 50 kHz or atleast 100 kHz. In some embodiments, the frequency of the lock-inamplifier AC voltage may be at most 0.05 Hz, at most 0.1 Hz, at most 0.5Hz, at most 1 Hz, at most 5 Hz, at most 10 Hz, at most 50 Hz, at most100 Hz, at most 500 Hz, at most 1 kHz, at most 5 kHz, at most 10 kHz, atmost 50 kHz or at most 100 kHz.

In some embodiments, the voltage of the AC signal generated may be atleast 0.1 V, at least 0.5V, at least 1V, at least 5V, at least 10V, atleast 20V, at least 50V, at least 100V, at least 500V, at least 1000V,at least 5 kV or at least 10 kV. In some embodiments, the voltage of thelock-in amplifier signal may be at most 0.1V at most 0.5V, at most 1V,at most 5V, at most 10V, at most 50V, at most 100V, at most 500V, atmost 1000V, at most 5 kV or at most 10 kV.

Imaging of separation channels: In some instances, the disclosed devicesand systems may be configured to perform imaging of all or a portion ofat least one separation channel to monitor a separation and/ormobilization reaction while it is performed. In some instances, thedisclosed devices and systems may be configured to perform imaging ofall or a portion of a plurality of separation channels to monitor aplurality of separation and/or mobilization reactions in parallel whilethey are performed. In some instances, separation and/or mobilizationreactions may be imaged using any of a variety of imaging techniquesknown to those of skill in the art. Examples include, but are notlimited to, ultraviolet (UV) light absorbance, visible light absorbance,fluorescence (e.g., native fluorescence or fluorescence resulting fromhaving labeled one or more analytes with fluorophores), Fouriertransform infrared spectroscopy, Fourier transform near infraredspectroscopy, Raman spectroscopy, optical spectroscopy, and the like. Insome instances the plurality of separation (or enrichment) channels maybe the lumens of a plurality of capillaries. In some instances, theplurality of separation (or enrichment) channels may be a plurality offluid channels within a microfluidic device. In some instances, all or aportion of a separation (or enrichment) channel, a junction orconnecting channel that connects an end of the separation channel and adownstream analytical instrument or an electrospray orifice or tip, theelectrospray orifice or tip itself, or any combination thereof may beimaged. In some instances, the separation (or enrichment) channel may bethe lumen of a capillary. In some instances, the separation (orenrichment) channel may be a fluid channel within a microfluidic device.

The wavelength range(s) used for imaging and detection of separatedanalyte bands will typically depend on the choice of imaging techniqueand the material(s) out of which the device or portion thereof arefabricated. For example, in the case that UV light absorbance is usedfor imaging all or a portion of the separation channel or other part ofthe microfluidic device, detection at about 220 nm (due to a nativeabsorbance of peptide bonds) and/or at about 280 nm (due to a nativeabsorbance of aromatic amino acid residues) may allow one to visualizeprotein bands during separation and/or mobilization provided that atleast a portion of the device, e.g., the separation channel or a portionthereof, is transparent to light at these wavelengths. In someinstances, the analytes to be separated may be labeled prior toseparation with, e.g., a fluorophore, chromophore, chemiluminescent tag,or other suitable label, such that they may be imaged using fluorescenceimaging, UV absorbance imaging, or other suitable imaging techniques. Insome instances, e.g., wherein the analytes comprise proteins produced bya commercial manufacturing process, the proteins may begenetically-engineered to incorporate a green fluorescence protein (GFP)domain or variant thereof, so that they may be imaged usingfluorescence. In some instances, labeling proteins or other analytemolecules may be performed using an approach to ensure that the labelitself doesn't interfere with or perturb the analyte property on whichthe chosen separation technique is based.

In some instances, imaging (or data derived therefrom) may be used totrigger, e.g., a mobilization step or other transfer of separatedanalyte fractions or portions thereof from a first plurality ofseparation channels to another plurality of separation channels, or froma first plurality of separation channels to a plurality of channels thatare in fluid communication with the outlet ends of the first pluralityof separation channels. For example, in some instances the disclosedmethods may comprise injecting analyte mixtures into a microfluidicdevice containing a first plurality of separation channels and a secondplurality of separation channels. The first plurality of separationchannels may contain a medium configured to bind an analyte from thesample analyte mixture. Accordingly, when the sample analyte mixturesare loaded or injected into the device, e.g., a microfluidic device, atleast a fraction of the analyte in each sample analyte mixture may bebound to the matrix and/or impeded from flowing through the firstplurality of separation channels. For example, injecting the analytemixtures into the microfluidic device can effect a chromatographicseparation in the first plurality of separation channels. An eluent canthen be injected into the microfluidic device such that at least afraction of the analyte, if present, is mobilized from the media in eachseparation channel. In some instances, the first plurality of separationchannels may be imaged while the analyte is mobilized. In someinstances, imaging of the first plurality of separation reactions maycomprise whole column (e.g., whole channel) imaging and/or imaging aportion of the separation channels. In some instances, an electric fieldmay be applied to the second plurality of separation channels when theimaging detects that an analyte fraction is disposed at intersections ofthe first plurality of separation channels and the second plurality ofseparation channels such that the analyte fractions areelectro-kinetically injected into the second plurality of separationchannels. For example, in some instances, the first plurality ofseparation channels and the second plurality of separation channels mayform a series of T-junctions. In some instances, imaging may be used todetect when an analyte fraction (e.g., a fraction of interest) is at oneor more of the series of T-junctions. Applying the electric field canelectro-kinetically inject the analyte fraction of interest (and,optionally, not other analyte fractions that are not located at theseries of T-junctions) into the second plurality of separation channelfor a second stage of separation. In some instances, the electric fieldmay be applied independently to one or more of the second plurality ofseparation channels depending on whether or not an analyte fraction ofinterest is detected at one or more of the T-junctions.

In some instances, imaging may be performed during mobilization tomonitor the mobilization reaction. In some instances, the imaging systemused to monitor the separation reaction may also be used to monitor themobilization reaction. In some instances, only a portion of the channelor plurality of channels may be imaged to monitor the mobilizationreaction. In some instances, the entire channel or plurality of channelsmay be imaged, and only a portion of the imaged channel or plurality ofchannels may be used to monitor the mobilization reaction. For example,the channels may be imaged at a given sampling rate, and for each imagegenerated, the portion of the image corresponding to the distal end ofthe channel or channels may be used to generate a mobility chromatogram.The mobility chromatogram may provide information on, for example, theaverage absorbance of a certain pixel width (e.g., 8 pixels) as afunction of time. In some instances, the pixel width of the image usedto generate the mobility chromatogram (e.g., corresponding to the distalend of the channel) may comprise at least 1 pixel, at least 2 pixels, atleast 3 pixels, at least 4 pixels, at least 5 pixels, at least 6 pixels,at least 7 pixels, at least 8 pixels, at least 9 pixels, at least 10pixels, at least 15 pixels, at least 20 pixels, at least 25 pixels, atleast 30 pixels, at least 35 pixels, at least 40 pixels, at least 50pixels, at least 60 pixels, at least 70 pixels, at least 80 pixels, atleast 90 pixels, at least 100 pixels.

The mobility chromatogram may be used to determine a parameter of themobilization reaction. For example, the mobility chromatogram may beused to calibrate the mass spectrometer, to determine the time-of-flightinformation, peak width, peak velocity, peak mobility, peak position,etc. of one or more analytes. In some instances, the mobilitychromatogram may be generated in real-time. In some instances, themobility chromatogram may be generated at a sampling rate (e.g., Nyquistsampling rate, 1-2 Hz, or a frequency that matches the sampling rate ofthe mass spectrometer). In some instances, the chromatogram may be usedto yield information on the absorbance of a segment of the channel as afunction of time.

Dynamic light scattering: In some instances, the systems and methods ofthe present disclosure comprise one or more detection methods comprisingdynamic light scattering (DLS). In some instances, DLS may be used toprovide information of the analytes, e.g., determine the sizedistribution profile of the separated analytes, the aggregation of theanalytes, the hydrodynamic radius of the analytes, etc. in at least oneseparation channel. DLS may be performed prior to, during, or followingthe separation of the analytes. DLS may be used in conjunction with oneor more of the methods described herein, e.g., for interlaced detectionof sample separation, mobilization, and/or analyte size profile. Forexample, DLS may be used in addition to imaging of the channel orchannels during one or more processes described herein (e.g. separation,mobilization, ejection).

System and system components: In some instances, the systems of thepresent disclosure may comprise one or more of the disclosed devices(e.g., microfluidic devices), one, two, three, four, or more highvoltage power supplies (or a single, multiplexed high voltage powersupply that allows independent control of two or more channels), anautosampler and/or fluid handling system, a fluid flow controller, animaging module, a dynamic light scattering module, a microplate-handlingrobotics module, a waste management module (e.g., to remove or preventaccumulation of fluid droplets from accumulating on the exterior of anelectrospray tip), an electrode interfacing unit, a processor orcomputer, or any combination thereof.

High voltage power supplies: In some instances, the two or more highvoltage power supplies of the disclosed systems (or a single,multiplexed high voltage power supply that allows independent control oftwo or more channels) are configured to provide simultaneous,independent electrical control of a plurality of separation channels,e.g., to simultaneously and independently apply a specified voltage orcurrent to each of a plurality of separation channels or auxiliary fluidchannels (e.g., mobilization channels used to deliver a chemicalmobilization agent to a separation channel following completion of anisoelectric focusing reaction). In some instances, the two or more highvoltage power supplies of the disclosed systems (or a single,multiplexed high voltage power supply that allows independent control oftwo or more channels) are configured to monitor and/or record thecurrent flowing through each separation channel of a plurality ofseparation channels (not just the total current). As described herein,the separation channels may comprise different samples or the samesample (e.g., aliquots of a sample). In some instances, the currentflowing through each separation channel may be used, for example, todetermine when an isoelectric focusing reaction is complete and/or todetect a failure (e.g., introduction or formation of a bubble in aseparation channel).

In some instances, the system may comprise two independent high voltagepower supplies, three independent high voltage power supplies, fourindependent high voltage power supplies, five independent high voltagepower supplies, six independent high voltage power supplies, sevenindependent high voltage power supplies, eight independent high voltagepower supplies, nine independent high voltage power supplies, tenindependent high voltage power supplies, eleven independent high voltagepower supplies, twelve independent high voltage power supplies, thirteenindependent high voltage power supplies, fourteen independent highvoltage power supplies, fifteen independent high voltage power supplies,sixteen independent high voltage power supplies, seventeen independenthigh voltage power supplies, eighteen independent high voltage powersupplies, nineteen independent high voltage power supplies, or twentyindependent high voltage power supplies. In some instances, the two ormore high voltage power supplies may be integrated or packaged as asingle multiplexed high voltage power supply that provides forsimultaneous and independent control of voltage and/or current for eachof a plurality of separation channels or auxiliary fluid channels (e.g.,mobilization channels used to deliver a chemical mobilization agent to aseparation channel following completion of an isoelectric focusingreaction).

In some instances, the two or more high voltage power supplies of thedisclosed systems are programmable, e.g., they may comprise an internalmicroprocessor and/or memory that allow the voltage and/or currentapplied to each of a plurality of separation channels or auxiliarychannels to be controlled by software downloaded to the high voltagepower supplies. In some instances, the two or more high voltage powersupplies of the disclosed systems may be configured for control by anexternal processor or computer.

In some instances, the two or more high voltage power supplies may beprogrammed or otherwise configured to run in constant voltage mode,e.g., where the voltage applied across each of a plurality of separationchannels and/or auxiliary channels is held fixed for the duration of aseparation reaction or for a specified period of time. In someinstances, the two or more high voltage power supplies may be programmedor otherwise configured to make stepwise changes in the voltage appliedacross each of a plurality of separation channels and/or auxiliarychannels from a first specified voltage to at least a second specifiedvoltage at one or more specified times. In some instances, the two ormore high voltage power supplies may be programmed or otherwiseconfigured to make two, three, four, five, or more than five stepwisechanges in voltage over the course of a separation reaction.

In some instances, the two or more high voltage power supplies may beprogrammed or otherwise configured to run in constant power mode, e.g.,to raise the voltage applied to a given separation channel as thecurrent drops during a separation reaction due to conductivity changes,thereby allowing one to increase the voltage to minimize separation timewithout inducing excess Joule heating.

As noted above, in some instances the electric field used to performelectrophoretic separation or isoelectric focusing reactions (or otherelectrokinetic injection or separation processes) may range from about 0V/cm to about 1,000 V/cm. Accordingly, in some instances, the two ormore high voltage power supplies of the disclosed systems may beconfigured to provide an adjustable voltage ranging from about 0 voltsto about 5,000 volts (e.g., for a 5 cm long separation channel). In someinstances, the two or more high voltage power supplies may be configuredto provide an adjustable voltage of at least 0, at least 5, at least 10,at least 50, at least 100, at least 500, at least 1,000, or at least5,000 volts. In some instances, the two or more high voltage powersupplies may be configured to provide an adjustable voltage of at most5,000, at most 1,000, at most 500, at most 100, at most 50, at most 10,or at most 5 volts. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the two or more high voltagepower supplies may be configured to provide an adjustable voltageranging from about 100 volts to about 1,000 volts. Those of skill in theart will recognize that the two or more high voltage power supplies maybe configured to provide an adjustable voltage of any value within thisrange, e.g., about 1,250 volts.

Electrode interfacing unit/fixture: In some instances, the disclosedsystems may comprise one or more fixtures, which may include electrodeinterfacing units (e.g., high voltage electrode interface units orfixtures) that are configured to interface the one or more electrodeswith one or more components of the system (e.g., one or more inlets ofthe microfluidic device). As described herein, the disclosedmicrofluidic devices may comprise two or more integrated electrodesconfigured to apply a voltage gradient along a separation channel orinterconnecting channel that intersects a separation channel. Theelectrodes may be integrated with or configured to interface with aplurality of inlet ports, outlet ports, sample and/or reagentintroduction channels, interconnecting channels, sample and/or reagentwaste channels, reservoirs (e.g., sample reservoirs, reagent reservoirs,or waste reservoirs), micropumps, microvalves, vents, traps, filters,membranes, and the like, or any combination thereof. In some instances,the fixture may comprise one or more membranes that allow for electricaland/or fluidic communication of the electrode and the microfluidicdevice. In some cases, the membrane may be in fluid and/or electricalcommunication with one or more reservoirs (e.g., the anolyte reservoiror catholyte reservoir) and/or the microfluidic device. In someinstances, the membrane may be coupled to the microfluidic device. Insome instances, the membrane may be used to prevent the introduction ofbubbles to the microfluidic device (e.g., channels or inlets) whenestablishing fluidic communication of the device with the one or morereservoirs. Alternatively, or additionally, the membrane may be used toprevent further introduction of bubbles, e.g., bubbles formed byelectrolysis at the electrodes, into the microfluidic device. In someinstances, the volume of an electrode reservoir (e.g., any reservoirwith which an electrode makes electrical contact) within the fixture maybe sufficiently large to minimize or eliminate pH changes in the buffercontained therein due to electrolysis at the electrode. In someinstances, the geometry of the fixtures may be configured to positionthe membrane to establish fluidic and/or electrical communication withthe microfluidic device. The membrane and/or electrode reservoir may bepositioned adjacent (e.g., on, next to, coupled to, orthogonal to, etc.)to the microfluidic device. In some cases, the membrane may be coupledto the microfluidic device (e.g., via a fitting mechanism). In someinstances, the geometries of the fixture may be employed to preventbending, folding, or nonplanar movement or configurations of themembrane, e.g., to prevent the formation of bubbles or application ofhydrodynamic pressure upon interfacing of the membrane with the device.For example, the fixture may comprise an insert, e.g., U-shapedstructure, where the membrane may be placed at the bottom (e.g., flatportion) of the U-shaped structure. In such an example, the U-shapedstructure may be coupled to the reservoir (e.g., an electrode reservoir,which can interface with the electrodes) and allow fluid communicationwith the membrane and the microfluidic device. The arms of the U-shapedstructure may comprise an inlet fluid path and an outlet fluid path. Inanother example, the fixture may comprise an insert, e.g., a Y-shapedstructure, where the membrane may be placed at the top of the Y-shapedstructure. In such an example, the Y-shaped structure may comprise thereservoir (e.g., electrode reservoir) and allow fluid communication withthe membrane and the microfluidic device.

In some instances, the membranes interface with the device via theoutlet fluid path and a port (e.g., the flat portion of the U-shapedstructure). At least one dimension of the port may take on a variety ofgeometries and be configured to prevent bending, folding, etc. of themembrane. For example, the port may be circular and may have a diameterof at most about 5 mm, at most about 4 mm, at most about 3 mm, at mostabout 2 mm, at most about 1 mm, or at most about 500 μm. The membranemay cover all or a portion of the port and may comprise any usefuldimension; for instance, the membrane may have a cross-sectional areathat is about 0.001 square millimeters (mm²), about 0.005 mm², about0.01 mm², about 0.05 mm², about 0.1 mm², about 0.5 mm², about 1 mm²,about 5 mm², about 10 mm², about 50 mm², about 100 mm², or about 500mm². The membrane may comprise a cross-sectional area that is at least0.001 (mm²), at least 0.005 mm², at least 0.01 mm², at least 0.05 mm²,at least 0.1 mm², at least 0.5 mm², at least 1 mm², at least 5 mm², atleast 10 mm², at least 50 mm², at least 100 mm², or at least 500 mm². Insome instances, the membrane may comprise a cross-sectional area that isat most 500 mm², at most 100 mm², at most 50 mm², at most 10 mm², atmost 5 mm², at most 1 mm², at most 0.5 mm², at most 0.1 mm², at most0.05 mm², at most 0.01 mm², at most 0.005 mm², or at most 0.001 mm². Themembrane may comprise a cross-sectional area that is in a range ofareas, e.g., between about 0.001 mm² and about 100 mm².

In some instances, the fixture may comprise an electrode reservoir, aninlet fluid channel comprising a first end and a second end, an outletfluid channel comprising a first end that is fluidically coupled to thesecond end of the inlet fluid channel, and a second end that isfluidically coupled to a separation channel (e.g., in a microfluidicdevice), which inlet fluid channel and the outlet fluid channelintersect with and are fluidically coupled to each other at a plane thatdefines or is parallel to a surface of the electrode reservoir. Themembrane may be disposed within the electrode reservoir at or adjacentto the plane at which the inlet fluid channel and outlet fluid channelintersect, such that the membrane covers all or substantially all of anopening comprising the intersection of the inlet fluid channel and theoutlet fluid channel (see, e.g., FIGS. 13A-F). In instances where thefixture comprises an insert comprising an inlet fluid path and an outletfluid path, the membrane and/or inlet fluid path and outlet fluid pathmay be configured to facilitate substantially bubble-free wetting of asurface of the membrane when the electrode reservoir is filled (e.g.,with strong electrolytes, buffers, reagents, etc.).

The membrane may be selected by desired material properties. Forexample, the membrane may be selected for a desired pore size,hydrophilicity, hydrophobicity, amphophilicity, oleophilicity, chargedor uncharged, inertness, mechanical properties (e.g., rigidity,compliance, flexibility, toughness), etc. In some cases, the membranemay comprise natural or synthetic materials. The membrane may compriseone or more polymers. In a preferred embodiment, the membrane comprisescellulose or regenerated cellulose and is hydrophilic. In anotherpreferred embodiment, the membrane comprises a polymer, e.g.,polytetrafluoroethylene (PTFE). In cases where a polymer is used, thepolymer (e.g., PTFE) may be manufactured or treated to obtain usefulproperties (e.g., woven, treated to render a surface hydrophilic, etc.).In another preferred embodiment, the membrane comprises a rigidmaterial, e.g., glass or ceramic. In some embodiments, the membrane maybe treated to be hydrophilic and/or uncharged.

In some instances, the membrane provides a high hydrodynamic resistanceand low electrical resistance connection between a high voltageelectrode positioned within the electrode reservoir and a fluid (e.g.,liquid, buffer, etc.) contained within the inlet fluid channel and theoutlet fluid channel. The hydrodynamic resistance between the reservoirand the intersection of the inlet fluid channel and outlet fluid channelmay be about 0.01 ((N/mm²)/(mm³/sec)), about 0.1 ((N/mm²)/(mm³/sec)),about 1((N/mm²)/(mm³/sec)), about 10 ((N/mm²)/(mm³/sec)), about 100((N/mm²)/(mm³/sec)), about 1,000 ((N/mm²)/(mm³/sec)), about 10,000((N/mm²)/(mm³/sec)), about 100,000 ((N/mm²)/(mm³/sec)), or about1,000,000 ((N/mm²)/(mm³/sec)). The hydrodynamic resistance between thereservoir and the intersection of the inlet fluid channel and outletfluid channel may be at least 0.01 ((N/mm²)/(mm³/sec)), at least 0.1((N/mm²)/(mm³/sec)), at least 1((N/mm²)/(mm³/sec)), at least 10((N/mm²)/(mm³/sec)), at least 100 ((N/mm²)/(mm³/sec)), at least 1,000((N/mm²)/(mm³/sec)), at least 10,000 ((N/mm²)/(mm³/sec)), at least100,000 ((N/mm²)/(mm³/sec)), or at least 1,000,000 ((N/mm²)/(mm³/sec)).The hydrodynamic resistance between the reservoir and the intersectionof the inlet fluid channel and outlet fluid channel may be at most1,000,000 ((N/mm²)/(mm³/sec)), at most 100,000 ((N/mm²)/(mm³/sec)), atmost 10,000 ((N/mm²)/(mm³/sec)), at most 1,000 ((N/mm²)/(mm³/sec)), atmost 100 ((N/mm²)/(mm³/sec)), at most 10 ((N/mm²)/(mm³/sec)), at most 1((N/mm²)/(mm³/sec)), at most 0.1 ((N/mm²)/(mm³/sec)), or at most 0.01((N/mm²)/(mm³/sec)). The hydrodynamic resistance between the reservoirand the intersection of the inlet fluid channel and outlet fluid channelmay be in a range of the values, e.g., between 1 ((N/mm²)/(mm³/sec)) and10,000 ((N/mm²)/(mm³/sec)).

In some instances, the hydrodynamic resistance of a portion of themembrane (e.g., a pore) may be calculated using the Hagen-Poiseuilleequation, equation 3:R _(Hydrodynamic)=8*viscosity*membrane thickness/(πr _(pore) ⁴)Where R_(Hydrodynamic) is the hydrodynamic resistance of one pore,viscosity is the viscosity of the bulk liquid, and r_(pore) is theradius of a single pore. The hydrodynamic resistance across the entiremembrane can then be equal to R_(Hydrodynamic) divided by the number ofpores. For example, if a membrane with 3 nm diameter pores and 100 μmthickness is used to inhibit hydrodynamic flow of an aqueous solution at25° C. (viscosity=0.89 cP), by this equation R_(Hydrodynamic) equals8*(0.89 cP)*(100 μm)/((π)*(1.5 nm)⁴), or, 4.48*10¹³ ((N/mm²)/(mm³/sec)).If the surface area of the membrane were 1 mm², and the pore areafraction was 5%, the number of pores equals to (1 mm²)*(0.05)/((n)*(1.5nm)²), or, 7.1*10⁹ pores, and the total hydrodynamic resistance equals(4.48*10¹³ ((N/mm²)/(mm³/sec)))/7.1*10⁹, or, 6330 ((N/mm²)/(mm³/sec)).

In some embodiments, the electrical resistance of a pore may becalculated using equation 4:R _(Electrical)=(solution resistivity)*(membrane thickness)/(πr _(pore)²)

Where r_(pore) is the radius of a single pore. For example, if thesolution resistivity is 500 (ohm)(cm), membrane thickness is 100 μm andpore diameter is 3 nm, then R_(Electrical) would be equal to (500ohm*cm)*(100 μm)/((π)*(1.5 nm)²), or, 7.1*10¹³ ohm. If the number ofpores were 7.1*10⁹ pores, the total electrical resistance of themembrane equals, in this example, 10000 ohm.

The electrical resistance between the electrode reservoir and theintersection of the inlet fluid channel and outlet fluid channel may beabout 10,000,000 ohms, about 1,000,000 ohms, about 100,000 ohms, about10,000 ohms, about 1,000 ohms, about 100 ohms, about 10 ohms, about 1ohm, about 0.1 ohm, or about 0.01 ohm. The electrical resistance betweenthe electrode reservoir and the intersection of the inlet fluid channeland outlet fluid channel may be at most 10,000,000 ohms, at most1,000,000 ohms, at most 100,000 ohms, at most 10,000 ohms, at most 1,000ohms, at most 100 ohms, at most 10 ohms, at most 1 ohm, at most 0.1 ohm,or at most 0.01 ohm. The electrical resistance between the electrodereservoir and the intersection of the inlet fluid channel and outletfluid channel may be in a range of values, e.g., between about 100,000ohms and 10,000,000 ohms.

In some instances, the ratio of the hydrodynamic resistance to theelectrical resistance between the electrode reservoir and theintersection of the inlet fluid channel and outlet fluid channel may beabout 0.001 ((N/mm²)/(mm³/sec))/Ohm, about 0.01 ((N/mm²)/(mm³/sec))/Ohm,about 0.1 ((N/mm²)/(mm³/sec))/Ohm, about 1 ((N/mm²)/(mm³/sec))/Ohm,about 10 ((N/mm²)/(mm³/sec))/Ohm, about 100 ((N/mm²)/(mm³/sec))/Ohm,about 1000 ((N/mm²)/(mm³/sec))/Ohm, about 10000 ((N/mm²)/(mm³/sec))/Ohm.In some instances, the ratio of the hydrodynamic resistance to theelectrical resistance between the electrode reservoir and theintersection of the inlet fluid channel and outlet fluid channel may beat least 0.001 ((N/mm²)/(mm³/sec))/Ohm, at least 0.01((N/mm²)/(mm³/sec))/Ohm, at least 0.1 ((N/mm²)/(mm³/sec))/Ohm, at least1 ((N/mm²)/(mm³/sec))/Ohm, at least 10 ((N/mm²)/(mm³/sec))/Ohm, at least100 ((N/mm²)/(mm³/sec))/Ohm, at least 1000 ((N/mm²)/(mm³/sec))/Ohm, atleast 10000 ((N/mm²)/(mm³/sec))/Ohm, or more. In some instances, theratio of the hydrodynamic resistance to the electrical resistancebetween the electrode reservoir and the intersection of the inlet fluidchannel and outlet fluid channel may be in a range of values, e.g.,between 1000 ((N/mm²)/(mm³/sec))/Ohm and 1,000,000((N/mm²)/(mm³/sec))/Ohm.

In some instances, the electrode reservoir may be filled with theelectrolyte solution at a concentration of about 0.1 millimolar (mM),about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 50 mM, about100 mM, about 500 mM, or about 1 molar (M). The electrolyte solutionconcentration may be at least 0.1 millimolar (mM), at least 0.5 mM, atleast 1 mM, at least 5 mM, at least 10 mM, at least 50 mM, at least 100mM, at least 500 mM, or at least 1 molar (M). The electrolyte solutionconcentration may be at most about 1M, at most about 500 mM, at mostabout 100 mM, at most about 50 mM, at most about 10 mM, at most about 5mM, at most about 1 mM, at most about 0.5 mM, or at most about 0.1 mM.The electrolyte solution concentration may be in a range ofconcentrations, e.g., between about 1 millimolar (mM) to about 500 mM.In some instances, during operation, the electrode reservoir is filledwith the electrolyte solution at a concentration between about 10 mM toabout 150 mM.

In some instances, during operation, the electrode reservoir is filledwith the electrolyte solution with a pH range between about 1.5 andabout 14. For instance, one electrode reservoir at a proximal end of aseparation channel may comprise an electrode solution of about 1.5 pHunits, and another electrode reservoir at a distal end of a separationchannel may comprise an electrode solution of about 14 pH units, or oneelectrode reservoir at a distal end of a separation channel may comprisean electrode solution of about 1.5 pH units, and another electrodereservoir at a proximal end of a separation channel may comprise anelectrode solution of about 14 pH units. It will be appreciated that thepH range or difference in pH between the electrode reservoirs can betuned based on a pH range that is useful for separating the analytespecies. For instance, if a mixture of analytes comprises expected pIvalues in a narrow pH range, the pH range or difference of the electrodereservoirs may be adjusted to be narrower in order to achieve higherseparation resolution for the given analyte mixture.

In some instances, the electrolyte solution comprises a strong acid, astrong base, or a highly soluble salt. Examples of strong acids include,but are not limited to, perchloric acid, hydrochloric acid, sulfuricacid, and the like. Examples of strong bases include, but are notlimited to, sodium hydroxide, potassium hydroxide, calcium hydroxide,and the like. Examples of highly soluble salts include, but are notlimited to, sodium chloride, potassium nitrate, magnesium chloride, andthe like. In some instances, a weak acid or weak base may be used as theelectrolyte solution. Examples of weak acids include, but are notlimited to, phosphoric acid, formic acid, acetic acid, carbonic acid,and the like. Examples of weak bases include, but are not limited to,ammonium hydroxide, diethylamine, dimethylamine, piperidine, and thelike. In some cases, the pH of the electrolyte solutions can be about1.5 pH units and about 14 pH units. In some cases, the pH of theelectrolyte solutions can be about 2 pH units and about 11 pH units. Insome cases, the pH of the electrolyte solutions can be about 3 pH unitsand about 9 pH units. In some cases, the pH of the electrolyte solutionscan be about 5 pH units and about 8 pH units.

Fluid flow controllers: In some instances, the disclosed systems maycomprise one or more programmable fluid flow controllers configured toprovide, e.g., independently-controlled, pressure-driven flow throughtwo or more separation channels (e.g., for use alone or in combinationwith a voltage gradient applied to the two or more separation channels)or auxiliary channels that intersect with the separation channels. Insome instances, pressure-driven flow may be used for mobilizingseparated analyte peaks out of a separation channel. In some instances,pressure-driven flow may be used, e.g., for introducing a chemicalmobilization agent into a separation channel (e.g., an electrolyte thatdisrupts the pH gradient used for isoelectric focusing), therebymobilizing separated analyte peaks out of the separation channel. Insome instances, pressure-driven flow may be used, e.g., for introducinga chemical mobilization agent into a separation channel (e.g., anelution buffer for eluting analytes from a stationary phase confinedwithin a separation channel), thereby mobilizing separated analyte peaksout of the separation channel. In some instances, the flow may becontrolled by integration of flow restrictors into the device, e.g.,long capillary or channel lengths to increase the hydrodynamicresistance and provide uniform flow profiles and electrosprayperformance.

Control of pressure-driven fluid flow through the disclosed devices andsystems will typically be performed through the use of pumps (or otherfluid actuation mechanisms) and valves. Examples of suitable pumpsinclude, but are not limited to, syringe pumps, programmable syringepumps, peristaltic pumps, diaphragm pumps, piston pumps and the like. Insome embodiments, fluid flow through the system may be controlled bymeans of applying positive pneumatic pressure at the one or more fluidinlets or sample or reagent reservoirs on the device. In someembodiments, fluid flow through the system may be controlled by means ofdrawing a vacuum at the one or more fluid outlets or waste reservoirs.Examples of suitable valves include, but are not limited to, checkvalves, electromechanical two-way or three-way valves, pneumatic two-wayand three-way valves, and the like. In some instances, one or moremicropumps or (e.g. peristaltic pumps, piezo pumps), microvalves (e.g.,metered injection valves, piezo valves, stopcock valves, slide valves)may be integrated within the device. In certain cases, control orpressure-driven fluid flow through the disclosed devices and systems maybe performed using a bladder, blister pack, pistons, screws, glassfrits, or a combination thereof. In some instances, the pressure-drivenfluid flow may be pulse-less.

In some embodiments, fluid flow through the system may be controlledusing one or more device or system parameters. In some instances, flowmay be generated in the device by altering the temperature of the system(e.g., to change the gas pressure in an area of the device) or byintroducing a temperature gradient. In some instances, the reservoirheight may be changed to drive flow through one or more channels of thedevice (e.g., via hydrostatic pressure). In some instances, a portion ofthe device (e.g., an inlet or outlet) may be exposed and allowed toevaporate, thereby driving fluid flow through the channels. In someinstances, the fluid flow may be pulse-less.

In some instances, fluid flow through the disclosed devices and systemsmay be performed electrically. For instance, electroosmotic flow in oneor more of the channels of the device or outside the channel may beperformed using, for example, an electroosmotic pump.

Different modes of fluid flow control may be utilized at differentpoints during the performance of the disclosed analyte separationmethods, e.g. forward flow (relative to the inlets and outlets for agiven device or separation channel), reverse flow, oscillating orpulsatile flow, or combinations thereof, may all be used. For example,in some instances, oscillating or pulsatile flow may be used, forexample, during device priming steps to facilitate dislodgement of anybubbles that may be trapped within the device. In some instances, thedevices may be subjected to vacuum (e.g., degassed) for device priming,e.g., to facilitate bubble-free introduction of a fluid or reagent.

Different fluid flow rates may be utilized at different points duringthe performance of the disclosed analyte separation methods. Forexample, in some instances of the disclosed devices and system, thevolumetric flow rate may vary from −100 ml/sec to +100 ml/sec. In someinstances, the absolute value of the volumetric flow rate may be atleast 0.001 ml/sec, at least 0.01 ml/sec, at least 0.1 ml/sec, at least1 ml/sec, at least 10 ml/sec, or at least 100 ml/sec. In some instances,the absolute value of the volumetric flow rate may be at most 100ml/sec, at most 10 ml/sec, at most 1 ml/sec, at most 0.1 ml/sec, at most0.01 ml/sec, or at most 0.001 ml/sec. The volumetric flow rate at agiven point in time may have any value within this range, e.g. a forwardflow rate of 2.5 ml/sec, a reverse flow rate of −0.05 ml/sec, or a valueof 0 ml/sec (i.e., stopped flow). In some instances, the pressure-drivenfluid flow mode and/or fluid flow velocities through each separationchannel and/or auxiliary fluid channels may be programmed independentlyof each other to follow a specified time-course.

Autosamplers and fluid handling systems: In some instances, thedisclosed systems may further comprise an autosampler or fluid handlingsystem configured for automated, independently controlled loading ofsample aliquots and/or other separation reaction reagents into aplurality of sample or reagent inlet ports to the separation channels.In some instances, a custom-built autosampler or fluid handling modulemay be incorporated into the disclosed systems. In some instances, acommercially-available autosampler or fluid handling module may beintegrated into the disclosed systems. Examples of suitablecommercially-available autosamplers include, but are not limited to, theAgilent 1260 Infinity Dual Loop Autosampler and 1260 Infinity HighPerformance Micro Autosampler (Agilent Technologies, Santa Clara,Calif.), the HT1500L HPLC Autosampler (HTA, Brescia, Italy), the SparkHolland Alias (Spark-Holland, Emmen, Netherlands), and the SIL-20A/ACHPLC Autosampler (Shimadzu, Columbia, Md.). Examples of suitablecommercially-available fluid handling systems (or liquid handlingsystems) include, but are not limited to, the Tecan Fluent® system(Tecan Trading AG, Switzerland), the Hamilton Microlab STAR and MicrolabNIMBUS systems (Hamilton, Reno, Nev.), and the Agilent Bravo AutomatedLiquid Handling Platform and Agilent Vertical Pipetting Station (AgilentTechnologies, Santa Clara, Calif.).

In some instances, one or more fluid flow controllers or fluid handlingsystems may be used for filling or replenishing one or more reservoirs.As described herein, the reservoirs may be in fluid communication with amembrane (e.g., comprised in a fixture and/or an electrode interfacingunit), which may interface with the microfluidic device and preventbubble formation at the device-membrane interface. The reservoirs may befilled using a variety of fluid controller or fluid handling systems.For example, a fluid controller may be used to distribute buffers orreagents to each reservoir. In some instances, the fluid controller maycomprise a pipette tip (e.g., a 1000 microliter pipette tip), and thereservoir may be configured to receive the pipette tip. In someinstances, the reservoir may comprise an access port to fill thereservoir from the bottom. In some instances, the reservoir may comprisea side port for access of the pipette tip without bubble formation. Insome instances, the reservoir may comprise a flange, which may aid inthe integration or interfacing of the fluid flow controller.

Waste management: In some embodiments, the system may further comprisewaste management modules, which may be integrated with (i.e., attachedto) or be separate from the microfluidic device. The waste managementmodule may be used to collect a waste product from the microfluidicdevice. In some instances, the waste management module may additionallyor alternatively be used to manage droplet formation at an outlet orsurface of the microfluidic device. For example, the waste managementmodule may be used to prevent droplets from forming at the outlet (e.g.,electrospray tip) of the device and/or wicking of the droplets to adifferent segment or portion of the device (e.g., the inlets, interfacedelectrodes, etc.). In some instances, the waste management module maycomprise application of positive or negative pressure (e.g., vacuum). Insuch cases, a vacuum may be applied to a part of the microfluidic device(e.g., the outlet or electrospray tip). For example, a flange or adaptormay be applied to the chip, thereby allowing the vacuum to be interfacedwith the device with minimal disruption to the placement of the deviceor to any downstream analysis units (e.g., mass spectrometer). Thevacuum may then be used to aspirate droplets or waste products as theyare expelled from the outlet or electrospray tip.

The vacuum may be applied through a variety of apparatus, which may beformed in one or more shapes. For example, the apparatus through whichthe vacuum is applied may be shaped like a horn or funnel. The horn orfunnel may be configured to apply a vacuum to the tip. In someinstances, the apparatus may be configured to swivel or move into adifferent position. In another embodiment, the vacuum may be appliedthrough a tube-shaped apparatus. The tube may be conical, cylindrical,or any other shape. In some instances, the tube may further comprise anopening module, which may be used to apply the vacuum and direct thewaste products to a waste receptacle. For example, the tube may beplaced between the chip (e.g., interfaced using a flange) and a massspectrometer, and the tube may comprise an opening module, e.g., vacuumtunnel that directs waste products from the electrospray tip, such thatthe waste product does not reach the mass spectrometer. In some cases,the tube may be oriented at an angle, e.g., perpendicularly, to theoutlet or electrospray tip. In such cases, the vacuum may be applied tothe tube and may aspirate droplets as the droplets exit the device. Insome instances, the tube may be transparent, such that one or moreimaging systems, as described herein, may be used to image theelectrospray tip. In another embodiment, the vacuum may be appliedthrough a modular device that may be configured to attach to a vacuum.For example, the modular device may be configured to clamp or attach toa portion of the device. Once the modular device is secured to thedevice, a vacuum may be applied to the modular device, thereby directingwaste products away from the microfluidic device.

In some instances, the waste management module may comprise the use ofpositive pressure. For example, an air knife may be used to directdroplets away from the electrospray tip. In such an example, the airknife may be connected to an air or nitrogen gas source and/orpressurizer to generate air (or nitrogen gas) pressure to eject thedroplets or direct the droplets away from the device or portion thereof(e.g., electrospray tip). In some instances, the waste management modulemay comprise a nebulizing unit. For example a nebulizer may beconfigured to secure to the chip. The nebulizer may comprise geometriesnecessary to direct air towards the chip such that the droplets or wasteproducts are directed away from the electrospray tip or outlet (e.g., toa waste receptacle). The nebulizer may comprise sealing mechanisms andmay be connected to an air source and/or pressurizer to generate airpressure to eject the droplets or direct the droplets away from theelectrospray tip. In some instances, the nebulizer may comprise anozzle. The nebulizer may be comprised of a polymer, metal, or ceramicmaterial.

In some instances, the waste management module may comprise the use ofmechanical approaches to remove waste and/or droplets from the outlet orelectrospray tip. For example, one or more wipers may be used tomechanically move (e.g., sweep) the droplets from the device.Alternatively, or additionally, an absorbent material may be integratedinto the waste management module, to absorb or wick away waste materialfrom the outlet or electrospray tip.

In some instances, the waste management module may be used inconjunction with other approaches for waste management. For example, thedevice may comprise a geometry or chemical/material properties thatallow for control of droplet formation at the outlet and/or to minimizewicking of droplets and fluids to a different segment or portion of thedevice (e.g., electrodes or inlets). In some instances, a coating may beused to allow for droplet formation at the tip or outlet of the deviceand may aid in the prevention of the wicking of fluids to other segmentsor portions of the device. In some cases, the coating may be ahydrophobic coating.

In some instances, the geometry or orientation of the device may be usedto control droplet formation at the outlet and/or to minimize wicking ofdroplets to a different segment or portion of the device. For example,the outlet or electrospray tip may be formed into a triangular tip toallow for optimal droplet formation. In some instances, the spatialorientation of the device may be used to control waste management. Forexample, the device may be angled such that an outlet (e.g., tip) isoriented downward, and the waste may be driven by gravity flow out ofthe microfluidic device. Any suitable angle may be used to direct thegravity flow out of the microfluidic device. For example, the angle maybe about 30°, 31°, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41°,42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, 50°, 51°, 52°, 53°, 54°, 55°,56°, 57°, 58°, 59°, 60°, etc. In some instances, the system furthercomprises a waste receptacle separate from the device for collecting thewaste product.

In some instances, the waste management module may obviate the need fora waste reservoir on the device. For example, the waste may be ejectedout of the device as droplets or a stream and may be removed, e.g., viaaspiration using a vacuum.

Imaging module: In some instances, the system may further comprise animaging module configured to acquire a series of one or more images ofthe two or more separation channels, or a portion thereof. In someinstances, the field-of-view of the images may comprise all or a portionof the two or more separation channels. In some instances, the imagingmay comprise continuous imaging of all or a portion of the two or moreseparation channels while separation and/or mobilization reactions areperformed. In some instances, the imaging may comprise intermittent orperiodic imaging of all or a portion of the two or more separationchannels while separation and/or mobilization reactions are performed.In some instances, the imaging may comprise acquiring UV absorbanceimages. In some instances, the imaging may comprise acquiringfluorescence images, e.g., of native fluorescence or fluorescence due tothe presence of exogenous fluorescent labels attached to the analytes.In some instances, the imaging module may be configured, for example, todetermine when an isoelectric focusing reaction is complete and/or todetect a failure (e.g., the introduction or formation of a bubble in aseparation channel).

Any of a variety of imaging systems or system components may be utilizedfor the purpose of implementing the disclosed methods, devices, andsystems. Examples include, but are not limited to, one or more lightsources (e.g., light emitting diodes (LEDs), diode lasers, fiber lasers,gas lasers, halogen lamps, arc lamps, etc.), condenser lenses, objectivelenses, mirrors, filters, beam splitters, prisms, image sensors (e.g.,CCD image sensors or cameras, CMOS image sensors or cameras), and thelike, or any combination thereof. In some instances, the one or morelight sources may comprise an array of light sources. For example, a LEDarray may be used to illuminate one or more regions of the device.Depending on the imaging mode utilized, the light source and imagesensor may be positioned on opposite sides of the microfluidic device,e.g., so that absorbance-based images may be acquired. In someinstances, the light source and image sensor may be positioned on thesame side of the microfluidic device, e.g., so that epifluorescenceimages may be acquired.

As noted above, images may be acquired continuously during theseparation and/or mobilization steps or may be acquired at random orspecified time intervals. In some instances, a series of one or moreimages are acquired continuously or at random or specified timeintervals. In some instances, a series of short exposure images (e.g.,10-20 images) are acquired on a fast (e.g., millisecond timescale) andare then averaged to provide a “single image” having improvedsignal-to-noise ratio. In some instances, a “single image” is acquiredevery 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, or atlonger time intervals. In some instances, longer exposure times may beused to improve signal-to-noise ratio. In some instances, the series ofone or more images may comprise video images.

Image processing: In some instances, as noted above, the system maycomprise processors, controllers, or computers configured to run imageprocessing software for detecting the presence of analyte peaks,determining the positions of pI markers or separated analyte bands,determining peak width, determining peak shapes (e.g., Gaussian fittingor other curve-fitting algorithms), or changes in any of theseparameters over time. In some instances, image processing may be usedfor detection of a failure, e.g., introduction or formation of a bubblein one of the two or more separation channels. Any of a variety of imageprocessing algorithms may be utilized for image pre-processing or imageprocessing in implementing the disclosed methods and systems. Examplesinclude, but are not limited to, Canny edge detection methods,Canny-Deriche edge detection methods, first-order gradient edgedetection methods (e.g., the Sobel operator), second order differentialedge detection methods, phase congruency (phase coherence) edgedetection methods, other image segmentation algorithms (e.g., intensitythresholding, intensity clustering methods, intensity histogram-basedmethods, etc.), feature and pattern recognition algorithms (e.g., thegeneralized Hough transform for detecting arbitrary shapes, the circularHough transform, etc.), and mathematical analysis algorithms (e.g.,Fourier transform, fast Fourier transform, wavelet analysis,auto-correlation, Savitzky-Golay smoothing, Eigen analysis, etc.), orany combination thereof.

Microplate-handling robotics: In some instances, the system may furthercomprise a microplate-handling robotics module configured to transportand replace microplates that serve as sources for samples and/orreagents. In some instances, the system may further comprise amicrofluidic device-handling robotics module configured to transport andreplace the microfluidic devices used in the system, e.g., after afailure is detected. In some instances, the microplate-handling and themicrofluidic device-handling may be handled by the same robotics module.In some instances, custom robotics may be incorporated into thedisclosed systems to perform these functions. In some instances,commercially-available robotics systems may be adapted and/or integratedinto the disclosed systems to perform these functions. Examples ofsuitable microplate handling robotics systems include, but are notlimited to, Tecan Robotic Gripper Arms (Tecan Trading AG, Switzerland)and the Agilent Direct Drive and BenchBot Robots (Agilent Technologies,Santa Clara, Calif.).

Failure mode detection and recovery: In some instances, the disclosedsystems may be configured for automated detection of system failures,e.g., current loss due to bubble introduction during sample loading orbubble formation during separation runs, incorrect current profile dueto incorrectly prepared samples, no current due to empty or underfilledwells in the sample plate. In some instances, the disclosed systems maybe configured to flag failures and automatically re-run samples forwhich a failure was detected in a corresponding separation channel. Insome instances, for example, the disclosed systems may be configured tore-load a specific sample from microtiter plate or other sample sourceand re-run the separation reaction.

Temperature control: In some instances, the disclosed systems andmethods may be subjected to temperature control. In some instances, aportion of system (e.g., a portion of the device) may be subjected totemperature control. In some instances, the system or one or morecomponents of the system may be cooled using, for example a Peltier, afan or other heat dissipater, an air knife. In some instances, thecooling system may be integrated with the waste management system (e.g.,air knife). In some instances, the cooling system may comprise acompressor for cooling. In some instances, the system may comprise anenvironmental or temperature-controlled chamber. In some instances,cooling blocks or pre-cooled blocks may be used (e.g., coupled to thestage or cartridge). In some instances, the system of component thereofmay be constructed from materials that allow for heat exchange with theenvironment. In some instances, the system may comprise a liquid heatexchanger.

Applications: The disclosed methods, devices, and systems have potentialapplication in a variety of fields including, but not limited to,proteomics research, cellular research, drug discovery and development,and clinical diagnostics. For example, the improved reproducibility andquantitation that may be achieved for separation-based characterizationof analyte samples using the disclosed methods may be of great benefitfor the characterization of biologic and biosimilar pharmaceuticalsduring development and/or manufacturing.

Biologics and biosimilars are a class of drugs which include, forexample, recombinant proteins, antibodies, live virus vaccines, humanplasma-derived proteins, cell-based medicines, naturally-sourcedproteins, antibody-drug conjugates, protein-drug conjugates and otherprotein drugs. The FDA and other regulatory agencies require the use ofa stepwise approach to demonstrating biosimilarity, which may include acomparison of the proposed product and a reference product with respectto structure, function, animal toxicity, human pharmacokinetics (PK) andpharmacodynamics (PD), clinical immunogenicity, and clinical safety andeffectiveness (see “Scientific Considerations in DemonstratingBiosimilarity to a Reference Product: Guidance for Industry”, U.S.Department of Health and Human Services, Food and Drug Administration,April 2015). Examples of the structural characterization data that maybe required for protein products include primary structure (i.e., aminoacid sequence), secondary structure (i.e., the degree of folding to formalpha helix or beta sheet structures), tertiary structure (i.e., thethree dimensional shape of the protein produced by folding of thepolypeptide backbone and secondary structural domains), and quaternarystructure (e.g., the number of subunits required to form an activeprotein complex, or the protein's aggregation state)). In many cases,this information may not be available without employing laborious,time-intensive, and costly techniques such as x-ray crystallography.Thus there is a need for experimental techniques that allow forconvenient, real-time, and relatively high-throughput characterizationof protein structure for the purposes of establishing biosimilaritybetween candidate biological drugs and reference drugs.

In some instances, the disclosed methods, devices, and systems may beused to provide structural comparison data for biological drugcandidates (e.g., monoclonal antibodies (mAb)) and reference biologicaldrugs for the purpose of establishing biosimilarity. For example, insome instances, determination of the isoelectric point for a drugcandidate and a reference drug may provide important evidence in supportof a demonstration of biosimilarity. In some embodiments, isoelectricpoint data for a drug candidate and a reference drug that have both beentreated with a site-specific protease under identical reactionconditions may provide important evidence in support of a demonstrationof biosimilarity. In some embodiments, the disclosed methods, devices,and systems may be used to monitor a biologic drug manufacturing process(e.g., to monitor bioreactor processes in real time) to ensure thequality and consistency of the product by analyzing samples drawn atdifferent points in the production process, or samples drawn fromdifferent production runs.

The disclosed devices and systems for performing multiple,independently-controlled separation reactions in parallel provide anumber of advantages over currently available technologies, for example,the ability to perform different isoelectric focusing reactions (orother separation reactions) in different channels (e.g., using differentpH gradients, different focusing times, different focusing voltages,etc.) for more detailed and accurate sample characterization (e.g., moreaccurate determination of pIs), or the ability to simultaneously processa plurality of samples in parallel using the same set of separationreaction conditions for higher throughput sample characterization.Furthermore, the independent monitoring and/or recording of currenttraces and/or voltage settings used for each separation channel may beadvantageous in meeting the data tracking requirements for FDAsubmissions when attempting to demonstrate biosimilarity, etc. As noted,in some instances, the disclosed devices and systems may be configuredto identify sample run failures, e.g., the presence or formation ofbubbles in the microfluidic device, and to initiate recovery steps,e.g., by automatically re-loading samples from a microtiter plate orother sample source and repeating the separation reaction.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1—Microfluidic Device Comprising Four Separation Channels

FIG. 1A provides a drawing of one non-limiting example of a microfluidicdevice for performing a plurality of separation reactions, e.g.,isoelectric focusing reactions. The device comprises a lower substrate101, which may be substantially planar, comprising fused silica in whichfluid channels measuring 210 microns wide and 100 microns in depth arefabricated using, e.g., embossing, laser micromachining, orphotolithography and wet chemical etching. The fluid channels are sealedby bonding substrate 101 to a transparent coverslip 102. In someinstances, e.g., in cases where UV absorbance imaging is used to monitorseparation and/or mobilization reactions, substrate 101 may befabricated from an optically transparent material. In some instances,e.g., where epifluorescence imaging is used to monitor separation and/ormobilization reactions, substrate 101 may be fabricated from anoptically opaque material. Although illustrated as a rectangular shape,it will be appreciated that the device may take any useful shape. Insome embodiments, the microfluidic device may comprise a tip (e.g., atthe distal end), which may allow for fluid to be directed away from thedevice (e.g., to a waste receptacle or analysis unit, e.g., massspectrometer).

Access to the fluid channels within the device is provided throughsample inlet ports 103, anode wells 104, cathode wells 106, sampleoutlet ports 107, and chemical mobilization agent inlet ports 109. Oneanode well 104 and cathode well 106 are in fluid- and electricalcommunication with a proximal end and distal end of each separationchannel 105, respectively (four separation channels are shown in thisnon-limiting example). The electrodes can, in some instances, be placedin contact with the anode well 104 and cathode well 106. The separationchannels extend beyond the cathode wells 106 to sample outlet ports 107(only labeled for two of the four separation channels shown in thefigure). Chemical mobilization agent inlet ports 109 are connected tothe distal ends of separation channels 105 via chemical mobilizationchannels 108 (only labeled for two of the four separation channels shownin the figure). As illustrated in FIG. 1A, the inlet ports 109 andoutlet ports 107 may be configured to be loaded through the side of thedevice, which may facilitate whole-channel or whole-device imaging.

For use in performing a plurality of isoelectric focusing reactions toseparate mixtures of proteins, protein samples are pre-mixed withampholyte pH gradient and pI markers before placing into vials andloading onto an autosampler. The samples are serially loaded into thedevice by the autosampler via the sample inlet ports 103 onto themicrofluidic device, through the separation channels 105, and out of thedevice to waste through the sample outlet ports 107.

A catholyte fluid (e.g., 1% N₄OH in H₂O) is loaded into cathode wells106, anolyte (e.g., 10 mM H₃PO₄) is loaded into the anode wells 104, anda mobilizer solution (e.g., 49% MeOH, 49% H₂O, 1% Acetic Acid) isconnected to mobilization agent inlet ports 109.

After all reagents are loaded, an electric field of, e.g., +600V/cm isapplied from one or more of the anode wells 104 to the correspondingcathode wells 106 by connecting electrodes to the anode wells 104 andcathode wells 106 to initiate isoelectric focusing. As noted above, thevoltages and/or currents applied to each of the separation channels 105may be controlled independently and may also be recorded as a functionof time. In some instances, the electrodes used for anodes and cathodesmay be integrated with the devices. For UV absorbance imaging, acollimated beam of light provided by a UV light source is aligned withthe separation channels 105, and an image sensor (e.g., a CCD camera orCMOS camera) is placed on the other side of the separation channels 105to measure the amount of light transmitted through each of theseparation channels 105, thereby imaging and detecting the focusedproteins (or other separated analytes) by means of their absorbance. Insome instances, the focused protein may be unlabeled and detectedthrough native absorbance at 220 nm, 280 nm, or any other wavelength atwhich the proteins will absorb light. For fluorescence imaging, i.e.,epifluorescence imaging, excitation light of a suitable wavelength isdelivered to the separation channels 105 by means of an optical assemblycomprising suitable dichroic reflectors and bandpass filters, andemitted fluorescence is collected from the separation channels 105 bythe same optical assembly and imaged onto the image sensor. In someinstances, focused proteins (or other separated analytes) may be imagedand detected using native fluorescence. In some instances, the focusedproteins may be detected using non-covalently bound fluorogenic,chromogenic, fluorescent, or chromophoric labels, such as SYPRO® Ruby,Coomassie Blue, and the like. In some instances, portions of the devicemay be constructed of an optically opaque material such that light mayonly be transmitted through the separation channels 105, thereby blockany stray light from reaching the image sensor without having passedthrough the separation channels 105 and increasing the sensitivity of UVabsorbance measurements.

Images of the focusing proteins in all or a portion of the separationchannels 105 can be captured continuously and/or periodically as theisoelectric focusing reactions are performed in the plurality ofseparation channels 105. In some instances, detection of the positionsof the pI markers in the images of the separation channels 105 may beused to determine the local pH as a function of position along theseparation channels and, by extrapolation, make more accuratedeterminations of pI for the separated proteins (or other analytes). Insome instances, when focusing is complete a positive pressure is appliedat sample inlet ports 103 and/or anode wells 104 to mobilize theseparated protein (or other analyte) mixture towards sample outlets 107.In some instances, when focusing is complete the electrodes connected tocathode wells 106 are disconnected, and electrodes in electricalcommunication with mobilizer channels 108 are used to apply an electricfield of 600V/cm from anode wells 104 to the chemical mobilization agentinlets 109 to electrophoretically introduce the mobilization agent intoseparation channels 105. In some instances, mild positive pressureapplied to mobilization agent inlets 109 may be used instead of, or inaddition to, electrophoretic introduction of a chemical mobilizationagent.

In the case of electrophoretic introduction of the mobilization agent,the acetic acid in the mobilizer solution is drawn by the electric fieldinto the separation channels 105, where it ionizes the proteins andampholytes and disrupts the pH gradient used for isoelectric focusing.The ionization of the enriched protein fractions causes them to migrateout of the separation channels 105 toward sample outlets 107. Continuingto image the separation channels 105 during the mobilization process canbe used to refine the determination of pI for each separated protein.

Example 2—Prophetic Example of the Use of the Disclosed Devices andSystems for Demonstration of Biosimilarity

One non-limiting example of the utility of the disclosed devices andsystems is in the field of biologics and the demonstration ofbiosimilarity. As noted above, the FDA and other regulatory agenciesrequire the use of a stepwise approach to demonstrating biosimilarity,which may include a comparison of the proposed product and a referenceproduct with respect to structure, function, animal toxicity, humanpharmacokinetics (PK) and pharmacodynamics (PD), clinicalimmunogenicity, and clinical safety and effectiveness. Examples of thestructural characterization data that may be required for proteinproducts include primary structure (i.e., amino acid sequence),secondary structure (i.e., the degree of folding to form alpha helix orbeta sheet structures), tertiary structure (i.e., the three dimensionalshape of the protein produced by folding of the polypeptide backbone andsecondary structural domains), and quaternary structure (e.g., thenumber of subunits required to form an active protein complex, or theprotein's aggregation state)). Accurate determination of proteinisoelectric points may provide an important datum for comparison ofbiologic drug candidates to a reference drug in order to demonstratebiosimilarity. Sample aliquots of a manufactured biosimilar candidateand a reference drug may be loaded into the disclosed devices or systemsand characterized under one or more sets of isoelectric focusingreaction conditions (e.g., using different buffers, pH gradients,applied voltages and/or currents, etc.) to determine accurate pI valuesunder the one or more sets of reaction conditions and provide valuablecomparison data for the biosimilar drug candidate and reference drug.Furthermore, the monitoring and recording of current traces for eachindividual separation reaction (and other operating parameters used forperforming the isoelectric focusing reactions) facilitates compliancewith FDA data submission requirements.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in any combination in practicing the invention.It is intended that the following claims define the scope of theinvention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

Example 3—Microfluidic Device Comprising Side Ports

FIG. 2 provides a schematic top-down view of one non-limiting example ofa microfluidic device for performing one or more separation reactions,e.g., isoelectric focusing reaction. The device comprises a substrate201, which may be substantially planar, in which fluid channelsmeasuring 210 microns wide and 100 microns in depth are fabricatedusing, e.g., embossing, laser micromachining, or photolithography andwet chemical etching. The fluid channels can be sealed by bonding thesubstrate 201 to a transparent coverslip (not shown). In some instances,e.g., in cases where UV absorbance imaging is used to monitor separationand/or mobilization reactions, substrate 201 may be fabricated from anoptically transparent material. In some instances, e.g., whereepifluorescence imaging is used to monitor separation and/ormobilization reactions, substrate 201 may be fabricated from anoptically opaque material.

Access to the fluid channels within the device is provided throughsample inlet ports 207, which may be located on the side of the chip.The chip may also comprise electrode reservoirs (e.g., anode wells 206,cathode wells 204), sample outlet ports 203, and chemical mobilizationagent inlet ports 209. One anode well 206 and cathode well 204 are influid- and electrical communication with a proximal end and distal end,respectively, of the separation channel 205. Chemical mobilization agentinlet ports 209 are connected to the distal ends of separation channels205 via chemical mobilization channels.

For use in performing a plurality of isoelectric focusing reactions toseparate mixtures of proteins, protein samples are pre-mixed withampholyte pH gradient and pI markers before placing into vials andloading onto an autosampler. The samples are serially loaded into thedevice by the autosampler via the sample inlet ports 207 onto themicrofluidic device, through the separation channels 205, and out of thedevice to waste through the sample outlet ports 203.

A catholyte fluid (e.g., 1% N₄OH in H₂O) is loaded into cathode wells204, anolyte (e.g., 10 mM H₃PO₄) is loaded into the anode wells 206, anda mobilizer solution (e.g., 49% MeOH, 49% H₂O, 1% Acetic Acid) isconnected to mobilization agent inlet ports 209. A membrane (not shown)may be interfaced with any of the anode or cathode wells (206 and 204)to provide electrical and fluid communication of the device with theelectrodes. An isometric cross-sectional schematic of the sample outletor ESI tip 203 is shown in FIG. 3.

Referring to FIG. 2, after all reagents are loaded, an electric fieldof, e.g., +600V/cm is applied from one or more of the anode wells 206 tothe corresponding cathode wells 204 by connecting electrodes to theelectrode reservoirs (anode wells 206 and cathode wells 204) to initiateisoelectric focusing. As noted above, the voltages and/or currentsapplied to each of the separation channels 205 may be controlledindependently and may also be recorded as a function of time. In someinstances, the electrodes used for anodes and cathodes may be integratedwith the devices. For UV absorbance imaging, a collimated beam of lightprovided by a UV light source is aligned with the separation channels205, and an image sensor (e.g., a CCD camera or CMOS camera) is placedon the other side of the separation channels 205 to measure the amountof light transmitted through each of the separation channels 205,thereby imaging and detecting the focused proteins (or other separatedanalytes) by means of their absorbance. In some instances, the focusedprotein may be unlabeled and detected through native absorbance at 220nm, 280 nm, or any other wavelength at which the proteins will absorblight. For fluorescence imaging, i.e., epifluorescence imaging,excitation light of a suitable wavelength is delivered to the separationchannels 205 by means of an optical assembly comprising suitabledichroic reflectors and bandpass filters, and emitted fluorescence iscollected from the separation channels 205 by the same optical assemblyand imaged onto the image sensor. In some instances, focused proteins(or other separated analytes) may be imaged and detected using nativefluorescence. In some instances, the focused proteins may be detectedusing non-covalently bound fluorogenic, chromogenic, fluorescent, orchromophoric labels, such as SYPRO® Ruby, Coomassie Blue, and the like.In some instances, portions of the device may be constructed of anoptically opaque material such that light may only be transmittedthrough the separation channels 205, thereby block any stray light fromreaching the image sensor without having passed through the separationchannels 205 and increasing the sensitivity of UV absorbancemeasurements.

Images of the focusing proteins in all or a portion of the separationchannels 205 can be captured continuously and/or periodically as theisoelectric focusing reactions are performed in the plurality ofseparation channels 205. In some instances, detection of the positionsof the pI markers in the images of the separation channels 205 may beused to determine the local pH as a function of position along theseparation channels and, by extrapolation, make more accuratedeterminations of pI for the separated proteins (or other analytes). Insome instances, when focusing is complete a positive pressure is appliedat sample inlet ports 207 and/or anode wells 206 to mobilize theseparated protein (or other analyte) mixture towards sample outlets 203.In some instances, when focusing is complete the electrodes connected tocathode wells 204 are disconnected, and electrodes in electricalcommunication with mobilizer channels 208 are used to apply an electricfield of 600V/cm from anode wells 206 to the chemical mobilization agentinlets 209 to electrophoretically introduce the mobilization agent intoseparation channels 205. In some instances, mild positive pressureapplied to mobilization agent inlets 209 may be used instead of, or inaddition to, electrophoretic introduction of a chemical mobilizationagent.

In the case of electrophoretic introduction of the mobilization agent,the acetic acid in the mobilizer solution is drawn by the electric fieldinto the separation channels 205, where it ionizes the proteins andampholytes and disrupts the pH gradient used for isoelectric focusing.The ionization of the enriched protein fractions causes them to migrateout of the separation channels 205. Continuing to image the separationchannels 205 during the mobilization process can be used to refine thedetermination of pI for each separated protein.

Example 4—Waste Management Using a Vacuum Apparatus

FIG. 4 provides a schematic of an example waste management system. Insome instances, the waste management system may be used to direct wasteaway from the microfluidic device 401. In some instances, the wastemanagement system may also be used to prevent wicking of droplets toanother portion of the device 401. As shown in FIG. 4, the device 401may be coupled to a stage 405. In some cases the microfluidic device 401may be inserted into a cartridge, which may then couple to the stage405. The stage and/or cartridge may comprise tubing and attachments toconnect fluidically and/or electrically to the device 401. The wastemanagement system may comprise a vacuum apparatus 407. The vacuumapparatus 407 may be shaped like a horn and may be configured to apply avacuum to the tip of the device 401. In some instances, the apparatus407 may be configured to attach to a portion of the device 401 using anadaptor, such as a flange 403. The flange 403 may comprise a slitthrough which a portion of the device 401 (schematically illustrated asa pointed tip) may fit. The vacuum apparatus 407 may be configured toswivel or move into a different position. For example, in a firstconfiguration, the apparatus 407 may be directed toward a wastereceptacle, thereby directing waste products to the waste receptacleupon application of the vacuum 409. In a second configuration, theapparatus 407 may be rotated, such that the sample or analyte may bedirected, for example, to a separate analysis unit, e.g., a massspectrometer.

In some instances, the stage 405 may be configured to move the device401. For example, the stage may allow for translation of the device 401in a direction that may be substantially parallel to one or morechannels of the device 401. In some instances, the stage 405 may allowfor translation of the device 401 in one or more directions. Forexample, the stage 405 may allow for translation of the device 401 in adirection that is substantially parallel to one or more channels of thedevice 401 as well as in a direction that is substantially perpendicularor orthogonal to one or more channels of the device 401. The stage 405may be configured to adjust the position of the device 401 such that thedevice 401 may be integrated with a downstream analysis unit (e.g., massspectrometer).

FIG. 5 provides a schematic of another example waste management system.Similar to the example shown in FIG. 4, the waste management system maybe used to direct waste away from the microfluidic device 501 and/or toprevent wicking of droplets to another portion of the device 501. Thedevice 501 may be coupled to a stage 505. In some cases, themicrofluidic device 501 may be inserted into a cartridge, which may thencouple to the stage 505. The stage and/or cartridge may comprise tubingand attachments to connect fluidically and/or electrically to the device501. The waste management system may comprise a vacuum apparatus 507.The vacuum apparatus 507 may be shaped like a cylinder and may beconfigured to apply a vacuum to the tip of the device 501. In someinstances, the apparatus 507 may be configured to attach to a portion ofthe device 501 using an adaptor, such as a flange 503. The flange 503may comprise a slit through which a portion of the device 501(schematically illustrated as a pointed tip) may fit. The vacuumapparatus 507 may be used to apply the vacuum 509 and direct the wasteproducts to a waste receptacle. For example, the vacuum apparatus 507may be placed between the chip (e.g., interfaced using a flange 503) anda mass spectrometer, and the vacuum apparatus 507 may comprise anopening module, e.g., vacuum tunnel 511 that directs waste products awayfrom the device 501, such that the waste product does not reach the massspectrometer. The vacuum 509 may be applied to the vacuum apparatus 507and may aspirate droplets as the droplets exit the device 501. In someinstances, the vacuum apparatus 507 may be transparent, such that one ormore imaging systems, as described herein, may be used to image theelectrospray tip.

In another embodiment, the vacuum may be applied through a modulardevice that may be configured to attach to a vacuum. For example, FIG. 6schematically shows another example of a waste management systemcomprising a clamp module 615. The clamp may be secured to the device601, and a vacuum 609 may be applied to the clamp device 615, therebydirecting waste products away from the microfluidic device. In someinstances, the device 601 may be coupled to the stage 605 or insertedinto a cartridge which may couple to the stage 605. In another example,FIG. 7 schematically shows another example of a waste management systemcomprising a tube vacuum apparatus 707. The tube may be positioned nearthe device 701, thereby directing waste products away from themicrofluidic device 701. A vacuum 709 may be applied to the tube vacuumapparatus 707 to direct the waste products away from the microfluidicdevice. In some instances, the tube may be positioned substantiallyorthogonal to the device 701, such that waste products can be directedaway from the microfluidic device without interfering with a downstreamanalysis unit, e.g., mass spectrometer. The device 701 may be coupled toor positioned on a stage 705.

Example 5—Waste Management Using Positive Pressure

In some instances, the waste management module may comprise the use ofpositive pressure. For example, in FIG. 8, an air knife 807 may be usedto direct droplets away from the device 801. In such an example, the airknife 807 may be connected to an air source and/or pressurizer togenerate air pressure to eject the droplets or direct the droplets awayfrom the device 801. In some instances, the system may further comprisea vacuum unit (not shown), which may be used to collect the dropletsthat are directed away from the device 801. Similar to FIGS. 4-7, thedevice 801 may be coupled to a stage 805 or may be coupled to acartridge which may couple to the stage 805.

In some instances, the waste management module may comprise a nebulizingunit. For example a nebulizer may be configured to secure to the chip.The nebulizer may comprise geometries necessary to direct air towardsthe chip such that the droplets or waste products are directed away fromthe electrospray tip or outlet (e.g., to a waste receptacle).

FIG. 9 schematically illustrates an example nebulizer 907 that may beconfigured to couple or secure to a device 901. The nebulizer 907 maycomprise a chamber and an inlet that may direct air or nitrogen gasinside the nebulizer 907. The nebulizer may additionally comprise slitsto direct the air or nitrogen gas out of the nebulizer (e.g., via anozzle, funnel, etc.). The nebulizer may be configured to direct air ornitrogen towards an outlet of the device 901 and thereby direct wasteproducts away from the device 901. In some embodiments, the nebulizer907 may be used to aerosolize the waste products.

FIGS. 10A-D schematically illustrate additional examples of designs fornebulizer 1007. The nebulizer 1007 may be configured to couple to orsecure to a device 1001. Similar to FIG. 9, the nebulizer 1007 maycomprise a chamber and an inlet that may direct air or nitrogen gasinside the nebulizer 1007. The nebulizer may additionally comprise slitsto direct the air or nitrogen gas out of the nebulizer (e.g., via anozzle, funnel, etc.). The nebulizer may be configured to direct airtowards an outlet of the device 1001 and thereby direct waste productsaway from the device 1001. In some embodiments, the nebulizer 1007 maybe used to aerosolize the waste products.

FIGS. 11A-D schematically illustrate another example nebulizer 1107. Thenebulizer 1107 may be configured to couple to or secure to a device1101. Similar to FIGS. 9-10, the nebulizer 1107 may comprise a chamberand an inlet that may direct air or nitrogen gas inside the nebulizer1107. The nebulizer may additionally comprise slits to direct the air ornitrogen gas out of the nebulizer (e.g., via a nozzle, funnel, etc.).The nebulizer may be configured to direct air towards an outlet of thedevice 1101 and thereby direct waste products away from the device 1101.In some embodiments, the nebulizer 1107 may be used to aerosolize thewaste products. The nebulizer 1107 may also comprise fasteners 1113,e.g., screws, to secure the nebulizer 1107 to the stage 1105, or incertain embodiments, to the cartridge (not shown).

Example 6—Fixture (Electrode Interfacing Unit) with Membrane

FIG. 12 schematically illustrates an example of a fixture 1200, such asthose described herein. The fixture 1200 may be configured to interfaceone or more electrodes with one or more components of the system (e.g.,one or more inlets of the microfluidic device). The electrodes may beconfigured to interface with a plurality of reservoirs 1203, which maybe in fluid and electrical communication with the device or a cartridgevia connections through valves 1205 comprising the device. Thereservoirs 1203 may comprise reagents and/or buffers for use in theseparation and/or mobilization reactions. In some instances, the fixture1200 may comprise one or more membranes (not shown) that allow forelectrical communication of the electrode and the microfluidic device orthe cartridge via connections through valves 1205. For instance, themembrane may be positioned at the bottom of each reservoir 1203, whichcan allow for fluidic and electrical communication of the reservoirs andelectrodes to the device or cartridge via connections through valves1205 and reduce the incidence of bubble formation at the interface ofthe reservoirs and the device or cartridge via connections throughvalves 1205.

In some instances, the geometry of the fixture may be configured toposition the membrane to establish fluidic and/or electricalcommunication with the microfluidic device. FIGS. 13A-F schematicallyillustrate a portion of the fixture 1300 comprising a membrane (notshown). In FIG. 13A, the portion of the fixture 1300 may comprise aninsert, e.g., U-shaped structure 1305 which may be connected to thereservoir 1303 and allow fluid and electrical communication with themembrane and the microfluidic device (not shown). The fixture 1300comprises an inlet fluid channel 1304 that is fluidically coupled to anoutlet fluid channel 1306, which is coupled to a separation channel (notshown). The inlet fluid channel 1304 and the outlet fluid channel 1306intersect at a plane 1308 that defines or is parallel to a surface ofthe reservoir 1303. At or adjacent to the plane 1308, a membrane (notshown) may be positioned. The membrane may cover all or substantiallyall of an opening comprising the intersection (e.g., plane 1308) of theinlet fluid channel 1304 and the outlet fluid channel 1306.

FIG. 13B illustrates schematically a view of the bottom of the portionof the fixture where the membrane may be positioned. FIG. 13C provides across section view of the insert comprising the U-shaped structure 1305.The U-shaped structure 1305 of the insert comprises an inlet fluid path1310 and an outlet fluid path 1312, which may facilitate substantiallybubble-free wetting of the membrane. FIG. 13D provides a schematic ofthe view of the bottom of the portion of the fixture. The membrane mayfluidically and/or electrically connect two ports 1307, which may beconnected (e.g., via the fluid inlet path 1310 and/or the fluid outletpath 1312) to the reservoir 1303 of fixture 1300. FIG. 13E providesanother view of the U-shaped structure 1305. The U-shaped structure 1305may comprise or be coupled to a membrane 1309. Fluidic and electricalcommunication with the reservoir 1303 and the membrane 1309 may beestablished using ports 1307 via the inlet fluid path 1310 and/or theoutlet fluid path 1312. FIG. 13F illustrates another view of theU-shaped structure 1305. The membrane 1309 may be coupled to orconnected to a port 1311 which may be connected to a channel 1315.Channel 1315 may comprise, in some instances, an inlet fluid channel(the region leading up to the U-shaped structure) and an outlet fluidchannel (the region following the U-shaped structure), which may connectto a microfluidic device or separation channel. The connection mayestablish fluidic and electric communication with the reservoir (notshown) and channel 1315.

Example 7—Reservoir Filling

FIG. 14 schematically illustrates an example method of providingreagents to one or more reservoirs 1403 of the systems described herein.The reservoirs 1403, which may be a part of the fixture (e.g., 1200 and1300), may be filled with buffers or reagents e.g., for the separationreaction and/or the mobilization reaction. In some instances, it may bedesirable to fill the reservoir from the bottom of the reservoir.Reagents and/or buffers may be introduced via an inlet fluid channel1404. In some instances, the reservoir 1403 may be configured to move(e.g., via translation) such that the reservoir 1403 may be moved to anupwards configuration, filled, and then resealed by moving the reservoir1403 back to the starting configuration. The outlet fluid channel 1406may be connected fluidically and/or electrically with a device orseparation channel (e.g., via a port and/or inlet or outlet fluidchannels).

In some instances, the reservoirs may be filled using conventionalmethods. FIG. 15 illustrates schematically an example method ofproviding reagents to one or more reservoirs 1503. In such an example apipette (e.g., a pipette, micropipette, etc.) 1519 may be used tointroduce buffers or reagents into the reservoir 1503. In someembodiments, the reservoir 1503 may further comprise a side port 1521.The introduction of buffers or reagents via the side port 1521 may helpin prevention of trapping bubbles on top of the membrane at the bottomof the reservoir 1503.

Example 8—Cartridge Design

FIG. 16 schematically illustrates an example system comprising acartridge, as described in certain embodiments herein. The cartridge1600 may comprise a microfluidic device 1601, which may comprise aplurality of inlet ports 1602. The ports 1602 may be in electrical andfluidic communication with ports 1623 which may be connected to a highvoltage power supply (e.g., via electrodes connected to a high voltagepower supply, which electrodes are connected to one or more reservoirs1603). The ports may be in fluidic and electrical communication with thereservoirs 1603 comprising reagents or buffers (e.g., anolyte,catholyte, mobilization agents, and background electrolyte). The flow ofthe reagents or buffers from the reservoirs 1603 can be controlled usingvalves 1625. In some cases one or more reservoirs 1603 may also beconnected to a restrictor 1627 (e.g., long tubing), which may stabilizethe flow rate and/or flow profile from the reservoir 1603 to the valve1625 to the port 1623. The reservoirs 1603 may each comprise a differentreagent or buffer; for example, one reservoir may comprise the anolytebuffer, another reservoir may comprise the catholyte buffer, and yetanother reservoir may comprise the mobilization buffer. The cartridge1600 or device 1601 may also be connected to a sample line, which may beused to supply the sample to the cartridge 1600 or device 1601 via avalve 1625.

FIGS. 17-20 show example embodiments of cartridges. In FIG. 17, thecartridge 1700 may comprise reservoirs 1703 which may be coupled to amembrane 1709, a tube 1711, and a plug 1713 which may seal the reservoirand comprise a hole, for example, for insertion of an electrode or forfilling the reservoirs. The cartridge 1700 may additionally comprise achannel 1718 for inserting and/or injecting the sample. The device 1701may be secured to the cartridge 1700 using stake features 1715. FIG. 18shows another example of a cartridge. An adaptor 1817 may be coupled tothe cartridge for facile filling of the reservoirs 1803. FIGS. 19A-Bshow another example of a cartridge comprising valves. In FIG. 19A, astopcock valve 1921 may be integrated with each reservoir 1903, whichmay allow for flow rate control. In FIG. 19B, a slider valve 1923 may beintegrated with each reservoir 1903. FIG. 20 shows another exampleembodiment of a cartridge. The device 2001 may be secured to thecartridge 2000 and fluidically and/or electrically connected toreservoirs (not shown) and/or the sample via several ports 2002 and/oran inlet fluid channel or outlet fluid channel. Fluidic connections maybe secured using gaskets 2030 or O-rings.

FIG. 21 schematically shows an example of securing features that may beused to secure the device to the cartridge. The cartridge 2100 maycomprise screws 2129 which may be used to fasten the device 2101 to thecartridge 2100. The cartridge 2100 may comprise one, two, three, or morescrews 2129. In some instances, nylon-tipped screws may be used. In someinstances, a pressure plate (e.g., a washer) may be added between thescrew 2129 and the device 2101, which may help evenly distribute stressand prevent stress concentrations of the device 2101 or cartridge 2100.

FIG. 22 shows an example schematic of securing features that may be usedto generate a fluidic seal of the cartridge to the device. The cartridge2200 may comprise screws 2229 which may be used to fasten the device2201 to the cartridge 2200. The cartridge 2200 may comprise one, two,three, or more screws 2229. In some instances, the cartridge 2200 maycomprise a gasket 2231. The gasket 2231 may interface with the device2201 and form a seal around an inlet or outlet port 2202 of the device2201. In some embodiments, the inlet or outlet ports 2202 may be securedto the cartridge 2200 using an O-ring.

FIG. 23 schematically shows electrical connection of one or morereservoirs to the device. The electrodes 2333 may comprise platinumwires and may be secured in place using, e.g., adhesives or otherfastening mechanisms, as described elsewhere herein. The electrodes 2333may be in contact with a reservoir 2303 thereby establishing electricalcommunication with the reservoir which may be in contact with themicrofluidic device, as described herein.

FIG. 24 schematically shows coupling of an instrument to the cartridge.The cartridge 2400 may be coupled to an instrument that may be used forproviding reagents to the reservoirs of the cartridge via fluid channels2435.

FIGS. 25-27 show additional example cartridges with varying reservoirstructures. FIG. 25 illustrates an embodiment where the cartridgecomprises a plurality of oblong reservoirs 2503. FIG. 26 illustrates anembodiment where the cartridge comprises an oblong reservoir 2603. Thereservoir 2603 may be skewed at an angle such that the reservoir 2603 ispositioned sufficiently far away from an outlet or tip of the device2601. The device 2601 may be connected to a high voltage reservoir 2605using a membrane 2609. In some instances, the membrane 2609 may bemechanically pressed between the high voltage reservoir 2605 and thedevice 2601. In some instances, the device 2601 may be sealed to one ormore fluid channels using a gasket 2631. FIG. 27 illustrates anembodiment where the cartridge comprises an additional manifold unit2735. The manifold unit may comprise the reservoirs 2703. The manifoldunit 2735 may be coupled to the cartridge using a fastening mechanismdescribed herein, e.g., screws and threads.

As described herein, the cartridges may comprise reservoirs, reagents,membranes, valves, securing devices or features (e.g., screws, pins(e.g., pogo pins), adhesives, levers, switches, grooves, form-fittingpairs, hooks and loops, latches, threads, clips, clamps, prongs, rings,rubber bands, rivets, grommets, ties, snaps, tapes, vacuum, seals),gaskets, o-rings, electrodes, or a combination thereof. The cartridgemay be monolithically built or may be modular and comprise removableparts. For instance, the microfluidic device may be configured to coupleremovably to the cartridge. Similarly, the reservoirs, membranes,valves, etc. may each be removable from the cartridge. In the case whereone or more components may be removable, the cartridge may be configuredsuch that each of the individual components may be aligned in place withsufficient tolerance by a user. For example, the cartridge may comprisegrooves and pins, such that the microfluidic device may be integrated bysliding the device along the cartridge until the cartridge reaches a pinfor alignment. In some instances, the device may be configured to bepositioned flush with the cartridge or a portion thereof. In someinstances, the device may be positioned into the cartridge such that oneor more inlets, outlets, etc. may be connected (e.g., fluidically and/orelectrically) to a reservoir, electrode, membrane and/or other usefulinterfacing unit. In some instances, the interfacing of the device andthe reservoirs, electrodes, etc. may be performed by a without anyadditional measurement or adjustment from the user. For example, thereservoirs may be configured to receive an electrode which snaps intoplace or is secured via a pogo pin, thereby establishing electricaland/or fluidic communication. It will be appreciated that these exampleconfigurations of the cartridge and device are not meant to be limiting,and that many different configurations of positioning the microfluidicdevice or other component of the cartridge may be achieved.

Example 9—Imaging Systems

FIGS. 28 A-D show different perspective views of an example imagingsystem disclosed herein. The imaging system may be used forwhole-channel imaging or whole-device imaging, or imaging of multiplechannels of a device. In some instances, the device may be secure to acartridge 2800. The cartridge may comprise a portion that istransparent. In some cases, the cartridge 2800 may be positioned near anillumination source, e.g., UV illuminator 2850. The device may beilluminated using the UV illuminator 2850 and light may be collected viaa detector 2857, e.g., a camera. In some instances, the light may bedirected to the detector 2857 using a mirror 2855, e.g., a turningmirror. The imaging system may further comprise a second detector 2859,which may comprise a camera that may be used to image electrospray. Insome instances, the imaging system may comprise an illumination ring2861. The device may be configured to direct the sample or analyte to adownstream analysis unit, e.g., a mass spectrometer via electrosprayionization, as described herein.

Example 10—System Configurations

FIGS. 29-31 schematically illustrate examples of systems describedherein. FIG. 29 illustrates an example of an instrument configured toperform one or more reactions described herein, e.g., separation ofanalytes via isoelectric focusing, mobilization of the analyte peaks,and downstream analysis via mass spectrometry. In certain embodiments,the system may comprise an auto-sampler 2901, which may be used toprocess and/or detect the sample, which may be located in the separationunit 2903, which may comprise the device used for the isoelectricfocusing, mobilization, etc. The system may comprise a compliantmechanism 2905 which may help with interfacing of the device in theseparation unit 2903 and a downstream analysis unit 2907. In someinstances, the downstream analysis unit 2907 is a mass spectrometer. Insome instances, the system may be situated on a motorized raising arm2909, which may be used to move any of the components described herein.For example, the raising arm 2909 may be used to raise and lower theauto-sampler 2901, the separation unit 2903, the compliant mechanism2905, and/or the analysis unit 2907 (e.g., mass spectrometer interfaceplate).

FIG. 30 illustrates another example of an instrument configured toperform one or more reactions described herein, e.g., separation ofanalytes via isoelectric focusing, mobilization of the analyte peaks,and downstream analysis via mass spectrometry. Similar to the system ofFIG. 29, the system may comprise an auto-sampler 3001, which may be usedto process and/or detect the sample, which may be located in theseparation unit 3003, which may comprise the device used for theisoelectric focusing, mobilization, etc. The system may comprise acompliant mechanism 3005 which may help with interfacing of the devicein the separation unit 3003 and a downstream analysis unit 3007. In someinstances, the downstream analysis unit 3007 is a mass spectrometer. Insome instances, the system may be situated on a motorized raising arm3009, which may be used to move any of the components described herein.For example, the raising arm 3009 may be used to raise and lower theauto-sampler 3001, the separation unit 3003, the compliant mechanism3005, and/or the analysis unit 3007 (e.g., mass spectrometer interfaceplate). In some cases, the system may also comprise one or more computeror computer processors 3011.

FIG. 31 illustrates an example of an instrument configured to performone or more reactions described herein, e.g., separation of analytes viaisoelectric focusing, mobilization of the analyte peaks, and downstreamanalysis via mass spectrometry. In certain embodiments, the system maycomprise an auto-sampler 3101, which may be used to process and/ordetect the sample, which may be located in the separation unit 3103,which may comprise the device used for the isoelectric focusing,mobilization, etc. A second system may be located adjacent or remotelyto the system and may comprise the separation unit 3103, a compliantmechanism 3105 which may help with interfacing of the device in theseparation unit 3103 and a downstream analysis unit 3107. In someinstances, the downstream analysis unit 3107 is a mass spectrometer. Insome cases, the system may also comprise one or more computer orcomputer processors 3111, which may be coupled to the auto-sampler 3101.

Example 11—Mobility Chromatograms

FIGS. 32A-B show example data of a mobilization reaction and mobilitychromatogram. Whole channel imaging may be performed during separation(e.g., via isoelectric focusing) of a sample comprising proteinisoforms. For example, a biologic therapeutic (e.g., antibodytherapeutic) may be separated using isoelectric focusing along a pHgradient (e.g., pH 5 to pH 10.5 gradient). Following the separationreaction, a mobilization reaction may be performed (e.g., to direct theseparated analytes to a downstream analysis unit, such as a massspectrometer). Whole-channel imaging of the mobilization reaction may beperformed over time, and a portion of each image may be used to generatea chromatogram. FIG. 32A illustrates the absorbance measurement of achannel of the device as a function of the pixel number (or distance)along the channel. Each pixel corresponds to approximately 25 micronsalong the length of the separation channel. FIG. 32B shows an examplemobility chromatogram generated by plotting the absorbance measurement(e.g., average absorbance) of a 3-pixel-wide section 3205 of the imageor absorbance plot of FIG. 32A as a function of time. In such anexample, plotting the 3-pixel-wide section 3205 can function as a pointdetector, thereby yielding information on the mobilization of theanalyte peaks as a function of time and allow for better correspondenceand/or validation of data obtained from the downstream analysis unit(e.g., mass spectrometer).

Example 12—Computer Systems

FIG. 33 shows an example software architecture system. The softwarearchitecture system may be integrated with the systems disclosed hereinand may comprise one or more computer processors. In some instances, theone or more computer processors may be configured to collect and/oranalyze data. The software architecture system may comprise a computerprocessing unit that comprises a controller service, which may be incommunication with a first in first out (FIFO) database. In someinstances, the FIFO database may be in communication with a secondcomputer processor, which may comprise a graphical user interface and aserver database. The second computer processor may be in communication,e.g., via cloud, with a customer database. In some instances, thecomputer processing unit may be in communication with one or morehardware units of the system (e.g., via a wired or wireless connection).For example, the computer processing unit may be connected via a USB hubto the stage, camera or cameras, high voltage power supplies,autosampler, flow control system (e.g., software and hardware formicrofluidic flow control, e.g., Fluigent Inc.) and/or other labequipment.

Example 13—Integrated Systems

FIG. 34 shows an example block diagram of an integrated system. Theintegrated system may comprise one or more systems disclosed herein. Thesystem may comprise an interfacing cartridge 3407, which may be influidic and/or electrical communication with a plurality of reservoirs3403. For example, the interfacing cartridge 3407 may be connected to ananolyte reservoir, a catholyte reservoir, a mobilizer reservoir, and anautosampler unit. Alternatively, or in addition to, the interfacingcartridge 3407 may be in fluidic and/or electrical communication with apressure control manifold 3405, which may be coupled to a fluid drivingmechanism, e.g., a pump. The interfacing cartridge 3407 may be coupledto a cartridge 3400 which may comprise the device 3401. The device 3401may be in electrical and/or fluidic communication with an anolyte highvoltage reservoir, a catholyte high voltage reservoir, a mobilizer highvoltage reservoir and a sample line. The anolyte high voltage reservoir,a catholyte high voltage reservoir, a mobilizer high voltage reservoirand a sample line may each be in fluidic and/or electrical communicationwith the interfacing cartridge 3407. The device 3401 may also be coupledto a waste management unit 3409, which may be used to direct waste awayfrom the device 3401 and, in some instances, also be used to direct thesample to the downstream analysis unit 3411. In some embodiments, thewaste management unit 3409 may comprise a nebulizer. In some instances,the downstream analysis unit 3411 may comprise a mass spectrometer.

The system may also comprise a plurality of imaging systems. Forexample, the system may comprise imaging system 3415, which may comprisea camera, an illuminator, a waste receptacle, and/or an adaptor, whichmay be used to interface with the analysis unit 3411. The system mayalso comprise imaging system 3417, which may comprise an illuminator(e.g., UV illumination source), a mirror, and/or a camera or othersuitable detector. In some instances, the detector (e.g., camera) may beconnected to a cooling source, e.g., fan or other temperature controlplatform.

FIG. 35 shows an example block diagram of an integrated system. Thesystem may comprise a sample 3501, a sample and reagent holder and/orprocessor 3503, which may be configured to store the samples and processthe samples (e.g., mix, add reagents, aspirate or dispense samples,etc.), a sample injector 3505, and a sample tip cleaner 3507. The sampletip cleaner may comprise mechanisms to wash the sample and/or thesystem. The system may also comprise a separation unit 3509, which maycomprise a cartridge comprising the device, an imaging system (e.g., UVilluminator and camera). The separation unit may be coupled to aplurality of controllers 3511 which may comprise fluid controls usingnegative pressure (e.g., vacuum) or positive pressure (e.g., rotary ordiaphragm pumps, valves, etc.). The controllers 3511 and/or theseparation unit 3509 may be coupled to a fluidics manifold 3513, whichmay comprise one or more reagent-containing reservoirs.

The separation unit 3509 may be used to perform a separation reaction(e.g., isoelectric focusing) and/or a mobilization reaction. Theseparation unit 3509 may be connected to or coupled to a communicationinterface 3515 (e.g., RFID), a high voltage power supply 3517, a wastemanagement unit 3519 (e.g., vacuum and waste receptacle), anotherimaging unit 3521, and/or a downstream analysis unit 3523 (e.g., a massspectrometer). In some instances, the separation unit 3509 may becoupled to a temperature control unit 3525. In some instances, one ormore systems described herein may comprise a temperature control unit3527 and/or other control unit, e.g., for instrument control 3529.

Example 14—Tracking Velocity of Analyte Peaks as they Leave theMicrofluidic Chip and Enter the Mass Spectrometer

Microfluidic channel network 100 in the device illustrated in FIG. 1B isfabricated in a 250-micron thick layer of opaque cyclic olefin polymer.Channel 112 is 250 microns deep, so it cuts all the way through the250-micron layer. All other channels are 50 microns deep. The channellayer is sandwiched between two transparent layers of cyclic olefinpolymer to fabricate a planar microfluidic device. Ports 102, 104, 106,108 and 110 provide access to the channel network for reagentintroduction from external reservoirs and electrical contact. Port 102is connected to a vacuum source, allowing channel 103 to act as a wastechannel, enabling the priming of the other reagents through the channelnetwork to “waste.” Acid (e.g., 1% formic acid) is primed through port108 to channels 109, 112, 114, and 103, and out to port 102. A sample(e.g., a peptide or protein diluted in 4% Pharmalyte 3-10, 12.5 mM pIstandard 3.38 (purified peptide, sequence: Trp-Asp-Asp-Asp), 12.5 mM pIstandard 10.17 (purified peptide, sequence: Trp-Tyr-Lys-Arg)) is primedthrough port 106 into channels 107, 112, 114, and 103 and out to port102. This leaves channel 112 containing the sample analyte. Base (e.g.,1% dimethylamine) is primed through port 104 into channels 105, 114, and103 and out to port 102. Mobilizer (e.g., 1% formic acid, 49% methanol)is primed through port 110 into channels 111, 114, and 103, and outchannel 103 to port 102.

Electrophoresis of the analyte sample in channel 112 is performed byapplying 4000V to port 108 and connecting port 110 to ground. Theampholytes in the analyte sample establish a pH gradient spanningchannel 112. Absorbance imaging of the separation is performed using a280 nm light source aligned to channel 112 and measuring thetransmission of 280 nm light through the channel 112 with a CCD camera.Software calculates the absorbance by comparing light transmissionduring separation or mobilization compared to a “blank” referencemeasurement taken in the absence of focused analyte before the analyteis run, then displays the absorbance per pixel over the length ofchannel 112. Locations where standards or analyte has focused aredisplayed as peaks in absorbance traces derived from the image data.

Once the analyte has completed focusing, a final focused absorbanceimage is captured. Software will identify the spatial position of the pImarkers and interpolate in between the markers to calculate the pI ofthe focused analyte fraction peaks. At this point, the control softwarewill trigger a relay disconnecting the ground at port 110, andconnecting port 104 to ground, as well as setting pressure on themobilizer reservoir connected to port 104 to establish flow of 100nL/min of mobilizer solution through port 104 into channels 105 and 114,and out of the chip at orifice 116. Orifice 116 is positioned 2 mm awayfrom a mass spectrometer ESI inlet, with an inlet voltage of −3500V to−4500V.

While the pressure driven flow directs mobilizer from port 104 toorifice 116, some of the formic acid in the mobilizer reagent willelectrophorese in the form of formate from channel 105, through channel112 to the anode at port 108. As the formate travels through channel 112it will disrupt the isoelectric pH gradient, causing the ampholytes,standards and analyte sample to increase charge and migrateelectrophoretically out of channel 112 into channel 114, where pressuredriven flow from port 110 will carry them into the ESI spray out oforifice 116.

While mobilization occurs, the software continues to capture absorbanceimages, and identifies peaks, tracking their migration out of theimaging channel 112 into channel 114. By tracking the time each peakexits imaging channel 112, its velocity, and the flow rate in channel114, the software can calculate the time the peak traverses channel 114and is introduced to the mass spectrometer via orifice 116, allowingdirect correlation between the original focused peak and the resultingmass spectrum.

Example 15—Microfluidic Device for Electrospray and Sample Processing

FIG. 2 provides a schematic top-down view of one non-limiting example ofa microfluidic device for performing one or more separation reactions,e.g., isoelectric focusing reaction. The device comprises a substrate201 in which fluid channels measuring 210 microns wide and 100 micronsin depth are fabricated using, e.g., embossing, laser micromachining, orphotolithography and wet chemical etching. The fluid channels can besealed by bonding the substrate 201 to a transparent coverslip (notshown). In some instances, e.g., in cases where UV absorbance imaging isused to monitor separation and/or mobilization reactions, substrate 201may be fabricated from an optically transparent material. In someinstances, e.g., where epifluorescence imaging is used to monitorseparation and/or mobilization reactions, substrate 201 may befabricated from an optically opaque material.

Access to the fluid channels within the device is provided throughsample inlet ports 207, which may be located on the side of the chip.The chip may also comprise anode wells 206, cathode wells 204, sampleoutlet ports 203, and chemical mobilization agent inlet ports 209. Oneanode well 206 and cathode well 204 are in fluid- and electricalcommunication with a proximal end and distal end of the separationchannel 205. Chemical mobilization agent inlet ports 209 are connectedto the distal ends of separation channels 205 via chemical mobilizationchannels.

For use in performing a plurality of isoelectric focusing reactions toseparate mixtures of proteins, protein samples are pre-mixed withampholyte pH gradient and pI markers before placing into vials andloading onto an autosampler. The samples are serially loaded into thedevice by the autosampler via the sample inlet ports 207 onto themicrofluidic device, through the separation channels 205, and out of thedevice to waste through the sample outlet ports 203.

A catholyte fluid (e.g., 1% N₄OH in H₂O) is loaded into cathode wells204, anolyte (e.g., 10 mM H₃PO₄) is loaded into the anode wells 206, anda mobilizer solution (e.g., 49% MeCN, 49% H₂O, 1% Formic Acid) isconnected to mobilization agent inlet ports 209. A membrane (not shown)may be interfaced with any of the anode or cathode wells (206 and 204)to provide electrical and fluid communication of the device with theelectrodes. An isometric schematic of the sample outlet or ESI tip 203is shown in FIG. 3.

Referring to FIG. 2, after all reagents are loaded, an electric fieldof, e.g., +600V/cm is applied from one or more of the anode wells 206 tothe corresponding cathode wells 204 by connecting electrodes to theanode wells 206 and cathode wells 204 to initiate isoelectric focusing.As noted above, the voltages and/or currents applied to each of theseparation channels 205 may be controlled independently and may also berecorded as a function of time. In some instances, the electrodes usedfor anodes and cathodes may be integrated with the devices. For UVabsorbance imaging, a collimated beam of light provided by a UV lightsource is aligned with the separation channels 205, and an image sensor(e.g., a CCD camera or CMOS camera) is placed on the other side of theseparation channels 205 to measure the amount of light transmittedthrough each of the separation channels 205, thereby imaging anddetecting the focused proteins (or other separated analytes) by means oftheir absorbance. In some instances, the focused protein may beunlabeled and detected through native absorbance at 220 nm, 280 nm, orany other wavelength at which the proteins will absorb light. Forfluorescence imaging, i.e., epifluorescence imaging, excitation light ofa suitable wavelength is delivered to the separation channels 205 bymeans of an optical assembly comprising suitable dichroic reflectors andbandpass filters, and emitted fluorescence is collected from theseparation channels 205 by the same optical assembly and imaged onto theimage sensor. In some instances, focused proteins (or other separatedanalytes) may be imaged and detected using native fluorescence. In someinstances, the focused proteins may be detected using non-covalentlybound fluorogenic, chromogenic, fluorescent, or chromophoric labels,such as SYPRO® Ruby, Coomassie Blue, and the like. In some instances,portions of the device may be constructed of an optically opaquematerial such that light may only be transmitted through the separationchannels 205, thereby block any stray light from reaching the imagesensor without having passed through the separation channels 205 andincreasing the sensitivity of UV absorbance measurements.

Images of the focusing proteins in all or a portion of the separationchannels 205 can be captured continuously and/or periodically as theisoelectric focusing reactions are performed in the plurality ofseparation channels 205. In some instances, detection of the positionsof the pI markers in the images of the separation channels 205 may beused to determine the local pH as a function of position along theseparation channels and, by extrapolation, make more accuratedeterminations of pI for the separated proteins (or other analytes). Insome instances, when focusing is complete a positive pressure is appliedat sample inlet ports 207 and/or anode wells 206 to mobilize theseparated protein (or other analyte) mixture towards sample outlets 203.In some instances, when focusing is complete the electrodes connected tocathode wells 204 are disconnected, and electrodes in electricalcommunication with mobilizer channels 208 are used to apply an electricfield of 675V/cm from anode wells 206 to the chemical mobilization agentinlets 209 to electrophoretically introduce the mobilization agent intoseparation channels 205. In some instances, mild positive pressureapplied to mobilization agent inlets 209 may be used instead of, or inaddition to, electrophoretic introduction of a chemical mobilizationagent.

In the case of electrophoretic introduction of the mobilization agent,the formic acid in the mobilizer solution is drawn by the electric fieldinto the separation channels 205, where it ionizes the proteins andampholytes and disrupts the pH gradient used for isoelectric focusing.The ionization of the enriched protein fractions causes them to migrateout of the separation channels 205. Continuing to image the separationchannels 205 during the mobilization process can be used to refine thedetermination of pI for each separated protein.

As the protein fractions and ampholytes migrate out of separationchannels 205 past cathode wells 204, they mix with mobilizer agent fromchannels 208 at intersection 210 (see FIG. 38). The mobilizer agent isbeing delivered to sample outlets 203 at a defined flowrate (e.g., 7.5nL/s), and this flowrate in channels 208 corresponds to a linearvelocity (e.g., 1.4 mm/s). As the enriched protein fractions andampholytes mix in the mobilizing agent, the new environment (reagent)causes a change in their electrophoretic mobility. This generates alinear electrophoretic velocity for ampholytes and protein fractionstowards the electrodes in electrical communication with mobilizerchannels 208—that is, in the opposite direction of the mobilizer linearvelocity toward the tip. In some instances, the chip network is designedso that the electrophoretic velocity of the enriched protein fractionswill be less than the mobilizer flow velocity, so that enriched proteinfractions migrate out the sample outlets (electrospray tips) 203 intoelectrospray and into a mass spectrometer for detection. In someembodiments, the chip network is designed so that the electrophoreticvelocity of some or all of the ampholytes is greater than the mobilizerlinear velocity, so that some or all of the ampholytes migrate towardsthe electrodes in electrical communication with mobilizer channels 208and are not introduced to the tips 203. In some instances, the ampholyteconcentration is diluted in this way to reduce amount of ionizablematerial in the electrospray, which can lead to improved ionization ofthe enriched protein fractions. In some instances, the channel networkmay be designed to maximize the introduction of the enriched proteinfractions in to the electrospray and to minimize the introduction ofother sample components into the electrospray. In some instances, thechannel network may be designed to maximize the introduction of theenriched protein fractions in to the electrospray and to minimize theintroduction of ampholytes into the electrospray.

For example, the electrophoretic mobility of NIST monoclonal antibody(NIST mAb) standard (pn 8671, NIST reference material) in 49% water, 1%formic acid, 50% MeCN mobilizer has been measured to be 1.5×10⁻⁴ cm²/Vs.In an electric field of strength 675V/cm, this would result in a linearvelocity of (1.5×10⁻⁴ cm²/Vs)×(675V/cm)=1.0×10⁻¹ cm/s, or, 1 mm/s towardthe electrode in electrical connection with mobilizer channels 208. Inthis example, if mobilizer channels 208 were etched to 50 microns deepby 110 microns wide, channels 208 would have a volume of 5.5 nL/mm, so amobilizer flowrate of 7.5 nL/s would correspond to a linear flowrate of1.4 mm/s. This would overcome the NIST mAb electrophoretic velocity of 1mm/s and the NIST mAb would exit the chip through sample outlet 203 intothe electrospray.

Pharmalyte brand ampholyte gradient pH 8-10.5 has been measured to havean electrophoretic mobility of on average of 2.7×10⁻⁴ cm²/Vs inmobilizer, which corresponds to a linear velocity average of 1.8 mm/s inour example 675V/cm electric field. In the example, channels 208 whichare 50 micron deep and 110 micron wide described above, this wouldovercome the 1.4 mm/s linear velocity of the mobilizer, and most of theampholyte would migrate towards the electrodes in electricalcommunication with mobilizer channels 208 and not exit the chip throughelectrospray tips 203, thus reducing the amount of ampholyte which couldinterfere with ionization of the enriched protein fractions in theelectrospray.

What is claimed is:
 1. A fixture comprising: an electrode reservoir; anda membrane disposed within the electrode reservoir, wherein the membraneis disposed at a surface of the electrode reservoir, wherein themembrane provides an electrical connection between an electrodepositioned within the electrode reservoir and a fluid contained withinat least one fluid channel in fluid communication with the electrodereservoir; wherein the electrode reservoir further comprises an insertdisposed within the electrode reservoir and positioned at or adjacent tothe membrane; wherein the insert comprises an inlet fluid path arm andan outlet fluid path arm, the inlet fluid path arm being in fluidiccommunication with the outlet fluid path arm; and the insert, the inletfluid path arm, and the outlet fluid path arm are integrally formed as aunitary structure.
 2. The fixture of claim 1, wherein the inlet fluidpath arm and the outlet fluid path arm facilitate substantiallybubble-free wetting of the surface of the membrane when the electrodereservoir is filled with a buffer solution.
 3. The fixture of claim 1,wherein the membrane comprises a first surface facing the electrodereservoir and a second surface facing the intersection of the inletfluid channel and the outlet fluid channel, wherein a hydrodynamicresistance between the first surface and the second surface is greaterthan 1 ((N/mm²)/(mm³/sec)).
 4. The fixture of claim 3, wherein anelectrical resistance between the first surface and the second surfaceis less than 10,000,000 ohms.
 5. The fixture of claim 1, wherein the atleast one fluid channel is in fluid communication with a separationchannel.
 6. The fixture of claim 5, wherein the separation channelcomprises a lumen of a capillary.
 7. The fixture of claim 5, wherein theseparation channel comprises a fluid channel within a microfluidicdevice.
 8. The fixture of claim 5, wherein the separation channel isconfigured to perform isoelectric focusing.
 9. The fixture of claim 5,wherein the separation channel is removably coupled to the at least onefluid channel.
 10. The fixture of claim 5, wherein the at least onefluid channel is coupled to the separation channel via a port, whereinthe port is disposed at an edge of a substrate comprising the separationchannel.
 11. The fixture of claim 1, wherein the membrane ishydrophilic.
 12. The fixture of claim 1, wherein the membrane comprisesa regenerated cellulose membrane.
 13. The fixture of claim 1, whereinthe membrane comprises a polymer.
 14. The fixture of claim 1, wherein across-sectional area of the membrane or opening is between about 0.001mm² and 100 mm².
 15. The fixture of claim 1, wherein the at least onefluid channel is fluidically coupled to a separation channel via avalve.
 16. The fixture of claim 1, wherein the electrode reservoircomprises an anolyte reservoir, a catholyte reservoir, or a mobilizationreagent reservoir.
 17. The fixture of claim 1, wherein the electrodereservoir has a dimension between about 10 micrometers and 2millimeters.
 18. The fixture of claim 1, wherein the electrode reservoircomprises a side port for introducing buffers or reagents.
 19. Thefixture of claim 1, wherein the insert is configured to facilitatewetting of a surface of the membrane.